We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.
Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.
Protein kinase RNA-activated (PKR), also known as eukaryotic initiation factor 2-alpha kinase 2 (EIF2AK2), is a well-characterized protein kinase that transmits information provided by RNAs. It belongs to the eukaryotic translation initiation 2 subunit alpha (eIF2α) kinase family and phosphorylates eIF2α at serine 51 in response to infection to suppress global translation1. In this context, PKR is activated by viral double-stranded RNAs (dsRNAs), which provide a platform for PKR dimerization and autophosphorylation2. In addition to eIF2α, PKR can also phosphorylate p53, insulin receptor substrate 1, inhibitor κB, and c-Jun N-terminal kinase (JNK) to regulate activity of numerous signal transduction pathways3,4,5,6.
PKR was originally identified as a kinase that phosphorylated eIF2α during poliovirus infection by recognizing poliovirus’ dsRNAs7,8. PKR is increasingly found to play multifaceted roles beyond immune response, and its aberrant activation or malfunction is implied in numerous human diseases. Activated/Phosphorylated PKR (pPKR) is frequently observed during apoptosis and is a common characteristic of patients with degenerative diseases, particularly neurodegenerative diseases such as Huntington’s, Parkinson’s, and Alzheimer’s disease9,10,11,12,13. In addition, PKR is activated under various stress conditions such as metabolic stress and heat shock14,15,16,17. On the other hand, inhibition of PKR results in increased cell proliferation and even malignant transformation18,19. PKR function is also important in normal brain function and during the cell cycle as the level of pPKR is elevated during the M phase20,21,22. In this context, pPKR suppresses global translation and provides cues to key mitotic signaling systems that are required for proper cell division20. Moreover, prolonged activation of PKR resulted in G2/M phase cell cycle arrest in Chinese hamster ovary cells23. Consequently, PKR phosphorylation is regulated by the negative feedback loop to ensure rapid deactivation during M/G1 transition21.
Despite the wide range of PKR function, our understanding of PKR activation is limited due to the lack of a standardized high-throughput experimental approach to capture and identify dsRNAs that can activate PKR. Previous studies have shown that PKR can interact with dsRNAs formed by two inverted Alu repeats (IRAlus)20,24, but the possibility of the existence of additional cellular dsRNAs that can activate PKR during the cell cycle or under stress conditions in human cells was unexplored. The conventional approach in identifying RNA-interactors of an RNA binding protein (RBP) uses UV light to crosslink RNA-RBP complexes25,26,27. A recent study applied this UV crosslinking approach in a mouse system and identified that small nucleolar RNAs can regulate PKR activation during metabolic stress16. By utilizing high crosslinking efficiency of formaldehyde, we presented an alternative method to identify PKR-interacting RNAs during the cell cycle in HeLa cells28. A similar approach has been applied to study other dsRBPs such as Staufen and Drosha29,30,31. We found that PKR can interact with various types of noncoding RNAs such as short interspersed nuclear element (SINE), long interspersed nuclear element (LINE), endogenous retrovirus element (ERV), and even alpha-satellite RNAs. In addition, we showed that PKR can interact with mitochondrial RNAs (mtRNAs), which form intermolecular dsRNAs through complementary interaction between the heavy-strand and the light strand RNAs28. A recent publication further supported our data that some mtRNAs exist in a duplex form and can activate dsRNA sensors such as melanoma differentiation-associated protein 5 to induce interferons32. More importantly, the expression and subcellular localization of mtRNAs are modulated during the cell cycle and by various stressors, which may be important in their ability to regulate PKR activation28.
In this article, we present a detailed protocol for a recently developed formaldehyde crosslinking and immunoprecipitation (fCLIP) method to capture and analyze PKR-interacting RNAs during the cell cycle. We demonstrate the method to prepare cell cycle arrest samples using thymidine and nocodazole. We then present the fCLIP process to isolate PKR-bound RNAs and a method to prepare high-throughput sequencing library to identify these RNAs. Furthermore, we delineate detailed procedures to analyze PKR-bound RNAs using qRT-PCR. Specifically, we present a strand-specific reverse transcription procedure to analyze the strandedness of mtRNAs. The described protocol is optimized for HeLa cells and PKR, but key steps such as the preparation of cell cycle sample, fCLIP, and strand-specific qRT-PCR analysis can be easily modified to study cellular dsRNAs or to identify RNA interactors of other dsRBPs.
1. Solution and cell preparation
2. Formaldehyde cross-linking and immunoprecipitation
3. Sequencing Library Preparation
4. Analysis of PKR-interacting RNAs using qRT-PCR
A schematic for the process to arrest HeLa cells at the S or M phase of the cell cycle is shown in Figure 1. For an M phase-arrested sample, we can clearly visualize round shaped cells under the microscope (Figure 2A). To examine the efficiency of the cell cycle arrest, the nuclear content of the cell can be analyzed using FACS (Figure 2B). Figure 3 shows representative data for immunoprecipitation efficiency test, where the D7F7 antibody shows a superior ability to immunoprecipitate PKR. This difference in the immunoprecipitation efficiency may have been reflected in the discrepancy in the class distribution of the high-throughput sequencing libraries prepared using two different PKR antibodies (Figure 4). The specificity of the D7F7 antibody is further confirmed using whole blot western analysis of the PKR immunoprecipitate (Figure 5A) and the total cell lysate (Figure 5B). Figure 6 shows the radioisotope signal before and after removal of rRNAs during high-throughput sequencing library preparation. Figure 7 shows the enrichment of mtRNAs in RNAs co-immunoprecipitated with PKR, but not in RNAs co-immunoprecipitated with rabbit IgG or DiGeorge syndrome chromosomal region 8 (DGCR8). Figure 8 shows the representative strand specific qRT-PCR analysis of PKR-bound mtRNAs in S or M-phase arrested samples.
Figure 1: Schematic for the preparation cell cycle arrest samples. (A, B) Schematics of the preparation of S (A) or M (B) phase-arrested HeLa cells. Please click here to view a larger version of this figure.
Figure 2: Analysis of cell cycle arrested samples. (A) Phase contrast images of S or M phase-arrested samples. Bars indicate 250 μm. (B) FACS analysis showing the nuclear content of the samples. Please click here to view a larger version of this figure.
Figure 3: Immunoprecipitation efficiency test for PKR antibodies. For successful enrichment of PKR-bound RNAs, an antibody with a definitive immunoprecipitation efficiency such as the D7F7 antibody should be used. Please click here to view a larger version of this figure.
Figure 4: RNA class distribution of sequencing libraries prepared using different PKR antibodies. Using different PKR antibodies for fCLIP resulted in a different class distribution of mapped sequencing reads. The discrepancy is likely due to the differences in the immunoprecipitation efficiency. This figure has been modified from Kim et al.28. Please click here to view a larger version of this figure.
Figure 5: Specificity of the D7F7 PKR antibody. (A, B) The whole blot western analysis of PKR immunoprecipitated (A) or total HeLa lysate (B) showed only one strong band corresponding to the size of PKR, indicating that the D7F7 antibody is highly specific in recognizing PKR. Please click here to view a larger version of this figure.
Figure 6: Radioisotope signal for PKR co-immunoprecipitated RNAs. (A) PKR co-immunoprecipitated RNAs could be detected by labeling their 5' ends with r-ATP. (B) Decrease in radioistope signal was observed upon successful rRNA removal. Please click here to view a larger version of this figure.
Figure 7: Validation of PKR-mtRNA interactions. Log2 fold enrichment of mtRNAs in RNAs co-immunoprecipitated with indicated antibodies. Only the PKR co-immunoprecipitated RNA sample showed strong enrichment of mtRNAs. Rabbit IgG and DGCR8 antibodies were used as negative controls. This figure has been modified from Kim et al.28. Please click here to view a larger version of this figure.
Figure 8: Strand-specific qRT-PCR analysis of PKR-bound mtRNAs. (A, B) Strand-specific reverse transcription was used to analyze the strandedness of mtRNAs that were co-immunoprecipitated with PKR for S (A) or M (B) phase-arrested cells. This figure has been modified from Kim et al.28. Please click here to view a larger version of this figure.
The process to prepare S or M phase-arrested samples is illustrated in Figure 1. To arrest cells at the S phase, we used a thymidine double block method where we treated cells with thymidine two times with a 9 h release in between to ensure high arrest efficiency (Figure 1A). For M phase arrest, we treated cells once with thymidine followed by a 9 h release and then applied nocodazole to block cells at prometaphase (Figure 1B). One key step in preparing the cell cycle sample is the release step after the first thymidine block. To completely remove thymidine, it is critical to wash the cells at least two times with fresh PBS. Improper washing and residual thymidine can result in increased heterogeneity and decreased cell viability. The success of M phase arrest can be confirmed visually based on the increase in the number of round-shaped cells (Figure 2A). However, the M phase sample still contains many interphase cells based on their morphology. To collect only the M phase arrested cells, we applied physical force to detach M phase cells from the surface. The nuclear content of the harvested cells was further examined using FACS, which showed a broad peak between 2n and 4n for the S phase and a sharp peak at 4n for the M phase-arrested sample (Figure 2B). The presented protocol is optimized for HeLa cells, which have a doubling time of approximately 24 h. The idea of double thymidine and thymidine-nocodazole block can be applied to other cell lines, but the exact drug treatment and release durations need to be optimized based on the doubling time of the target cells.
To identify PKR-interacting RNAs, we crosslinked PKR-RNA complexes with formaldehyde and enriched them through immunoprecipitation. A key factor that determines the accuracy of the subsequent analysis is the efficiency of immunoprecipitation. We have tested numerous antibodies that target different epitopes of the PKR protein in order to determine the antibody for the high-throughput sequencing library preparation. As shown in Figure 3, we found that the D7F7 antibody that recognizes the linker region between the dsRNA binding domains and the catalytic domain showed superior ability in capturing PKR. The other antibody shown in Figure 3 (Milli) recognizes the N-terminal region, but shows a poor ability in immunoprecipitating PKR. Consequently, the high-throughput sequencing library prepared using the Milli antibody contained many background sequencing reads that are dispersed throughout the genome, particularly in introns, without distinct accumulations at specific regions28 (Figure 4). We believe the discrepancies in the two sequencing libraries are mostly due to the differences in the antibodies’ abilities in capturing PKR during the immunoprecipitation step. We further examined the specificity of the D7F7 PKR antibody through whole blot western analyses of immunoprecipitate (Figure 5A) and total HeLa lysates (Figure 5B). In both blots, we only observed one strong band that corresponds to PKR. This indicates that the D7F7 antibody is highly specific and that the sequencing data obtained using the D7F7 antibody likely reflect true RNA interactors of PKR.
A critical step during the high-throughput sequencing library preparation is the removal of rRNAs. Since we crosslinked RNA-RBP complexes with formaldehyde, we used an ultrasonicator for complete lysis of the cells. This process resulted in fragmentation of rRNAs, which significantly reduced the efficiency of rRNA removal using the rRNA Removal Kit. To resolve this problem, we first used the rRNA Removal Kit followed by the rRNA Depletion Kit (see Table of Materials), which almost completely removed rRNAs and less than 1% of the total sequencing reads were mapped to rRNAs. We used these two kits sequentially because the rRNA Depletion Kit has a maximum capacity of only 1 μg while the rRNA Removal Kit has a maximum capacity of 5 μg. We have experienced that using more than the recommended amount of the total RNA results in significant amount of sequencing reads mapped to rRNAs. The successful removal of rRNA can be confirmed after labeling the RNAs with r-ATP through the PNK reaction. While the rRNA depleted RNAs showed a distinct band around 150 nt, the sample before rRNA removal shows strong signal throughout the region corresponding to the 50 ~ 300 nt (Figure 6).
One limitation of formaldehyde crosslinking is the decrease immunoprecipitation efficiency. Other applications of formaldehyde crosslinking such as immunocytochemistry typically use 4% paraformaldehyde solution for fixation. However, such a strong fixation condition cannot be applied for fCLIP experiment because it significantly decreases the immunoprecipitation efficiency, which results in a higher background. Moreover, formaldehyde fixation crosslinks protein-protein complexes in addition to protein-RNA complexes. Therefore, one needs to pay caution in interpreting the fCLIP data.
As reported previously, mtRNAs form intermolecular dsRNAs that are recognized by PKR28. We first validated our sequencing data by examining PKR-mtRNA interactions through qRT-PCR. We used rabbit IgG and DGCR8 antibodies as negative controls, which did not show any enrichment of mtRNAs (Figure 7). Of note, DGCR8 was used as a nuclear dsRNA binding protein that is physically separated from mtRNAs. At the same time, PKR co-immunoprecipitated RNAs showed strong enrichment of mtRNAs (Figure 7).
To further analyze PKR-mtRNA interactions, we performed strand-specific reverse transcription to distinguish the heavy-strand mtRNAs from the light-strand mtRNAs (Figure 8). We designed reverse transcription primers that have a CMV promoter sequence followed by a gene-specific sequence and then used a CMV promoter sequence as the left primer and gene-specific right primers for the qPCR analysis. We have also tested SP6 promoter and pGEX sequencing primer sequences instead of the CMV promoter sequence. We found that while CMV promoter and pGEX sequencing primer sequences showed good result, using the SP6 promoter sequence did not. The difference is due to the low GC content of the SP6 promoter sequence (~33%) compared to those of the CMV promoter (~67%) and the pGEX sequencing primer (~65%) sequences. The proposed scheme for strand-specific reverse transcription can easily be applied to other intermolecular dsRNAs, but when designing the reverse transcription primers, the GC content needs to be taken into consideration.
Overall, we demonstrated the preparation of cell cycle arrested samples and the enrichment of PKR-interacting RNAs through formaldehyde crosslinking and immunoprecipitation. Using a highly efficient D7F7 antibody, we have successfully isolated PKR-bound RNAs and identified these RNAs by generating and analyzing a high-throughput sequencing library. Furthermore, to analyze the strandedness of mtRNAs bound to PKR, we presented a strand-specific reverse transcription approach. We expect that the presented protocol can be easily optimized to study RNA interactors of other dsRBPs and strandedness of intermolecular dsRNAs that are formed via complementary interaction between sense and antisense transcripts.
The authors have nothing to disclose.
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korean government Ministry of Science and ICT (NRF-2016R1C1B2009886).
0.5 M EDTA, pH 8.0 | Thermo Fisher Scientific | AM9260G | |
1 M Tris, pH 7.0 | Thermo Fisher Scientific | AM9855G | |
1 M Tris, pH 8.0 | Thermo Fisher Scientific | AM9855G | |
1.7 mL microcentrifuge tube | Axygen | MCT-175-C | |
10% Nonidet-p40 (NP-40) | Biosolution | BN015 | |
10% Urea-acrylamide gel solution | 7 M (w/v) Urea and 0.5X TBE, stored protected from light at 4 °C | ||
10X DNA loading buffer | TaKaRa | 9157 | |
15 mL conical tube | SPL | 50015 | |
3' adaptor | 5'-rApp NN NNT GGA ATT CTC GGG TGC CAA GG/3ddC/-3' | ||
3 M Sodium Acetate pH 5.5 | Thermo Fisher Scientific | AM9740 | |
5' adaptor | 5'-GUU CAG AGU UCU ACA GUC CGA CGA UCN NNN-3' | ||
5 M NaCl | Thermo Fisher Scientific | AM9760G | |
50 mL conical tube | SPL | 50050 | |
Acid-phenol chloroform, pH 4.5 | Thermo Fisher Scientific | AM9722 | |
Agencourt AMPure XP | Beckman Coulter | A63881 | Magnetic beads DNA/RNA clean up |
Antarctic alkaline phosphatase | New England Biolabs | M0289S | |
Anti-DGCR8 | Made in house | ||
Anti-PKR (D7F7) | Cell signaling technology | 12297S | |
Anti-PKR (Milli) | Millipore EMD | 07-151 | |
ATP (100 mM) | GE Healthcare | GE27-2056-01 | |
Bromophenol blue sodium salt | Sigma-aldrich | B5525 | |
Calf intestinal alkaline phosphatase | TaKaRa | 2250A | |
Cell scraper 25 cm 2-position | Sarstedt | 83.183 | |
CMV promoter sequence | 5'-CGCAAATGGGCGGTAGGCGTG-3' | ||
Dulbecco's modified eagle medium | Welgene | LM001-05 | |
dNTP mixture (2.5 mM) | TaKaRa | 4030 | |
Ethanol, Absolute, ACS Grade | Alfa-Aesar | A9951 | |
Fetal bovine serum | Merck | M-TMS-013-BKR | |
Formamide | Merck | 104008 | |
Glycine | Bio-basic | GB0235 | |
GlycoBlue coprecipitant (15 mg/mL) | Thermo Fisher Scientific | AM9516 | |
Isopropanol | Merck | 8.18766.1000 | |
NEBNext rRNA Depletion Kit | New England Biolabs | E6318 | rRNA Depletion Kit |
Nocodazole | Sigma-Aldrich | M1404 | |
Normal rabbit IgG | Cell signaling technology | 2729S | |
Paraformaldehyde | Sigma-Aldrich | 6148 | |
PCR forward primer (RP1) | 5'-AAT GAT ACG GCG ACC ACC GCG ATC TAC ACG TTC AGA GTT CTA CAG TCC GA-3' | ||
PCR index reverse primer (RPI) | 5'-CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA-3' | ||
PCR tubes with flat cap, 0.2 mL | Axygen | PCR-02-C | |
Phosphate bufered saline (PBS) Tablet | TaKaRa | T9181 | |
Phusion high-fidelity DNA polymerase | New England Biolabs | M0530 | High-fidelity polymerase |
PlateFuge microcentrifuge with swing-out rotor | Benchmark | c2000 | |
Polynucleotide kinase (PNK) | TaKaRa | 2021A | |
Protease inhibitor cocktail set III | Merck | 535140-1MLCN | |
Proteinase K, recombinant, PCR Grade | Sigma-Aldrich | 3115879001 | |
qPCR primer sequence: CO1 Heavy | Forward/Reverse: 5′-GCCATAACCCAATACCAAACG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CO1 Light | Forward/Reverse: 5′-TTGAGGTTGCGGTCTGTTAG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CO2 Heavy | Forward/Reverse: 5′-CTAGTCCTGTATGCCCTTTTCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CO2 Light | Forward/Reverse: 5′-GTAAAGGATGCGTAGGGATGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CO3 Heavy | Forward/Reverse: 5′-CCTTTTACCACTCCAGCCTAG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CO3 Light | Forward/Reverse: 5′-CTCCTGATGCGAGTAATACGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CYTB Heavy | Forward/Reverse: 5′-CAATTATACCCTAGCCAACCCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CYTB Light | Forward/Reverse: 5′-GGATAGTAATAGGGCAAGGACG -3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: GAPDH | Forward/Reverse: 5′-CAACGACCACTTTGTCAAGC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND1 Heavy | Forward/Reverse: 5′-TCAAACTCAAACTACGCCCTG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND1 Light | Forward/Reverse: 5′-GTTGTGATAAGGGTGGAGAGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND4 Heavy | Forward/Reverse: 5′-CTCACACTCATTCTCAACCCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND4 Light | Forward/Reverse: 5′-TGTTTGTCGTAGGCAGATGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND5 Heavy | Forward/Reverse: 5′-CTAGGCCTTCTTACGAGCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND5 Light | Forward/Reverse: 5′-TAGGGAGAGCTGGGTTGTTT-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND6 Heavy | Forward/Reverse: 5′-TCATACTCTTTCACCCACAGC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND6 Light | Forward/Reverse: 5′-TGCTGTGGGTGAAAGAGTATG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
Random hexamer | Thermo Fisher Scientific | SO142 | |
Recombinant Dnase I (Rnase-free) (5 U/μL) | TaKaRa | 2270A | |
Recombinant Rnase inhibitor (40 U/μL) | TaKaRa | 2313A | |
Ribo-Zero rRNA Removal Kit | Illumina | MRZH116 | rRNA Removal Kit |
Rotator | FINEPCR, ROTATOR AG | D1.5-32 | |
RT primer sequence: CO1 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGTTGAGGTTGCGGTCTGTTAG-3′ | ||
RT primer sequence: CO1 Light | 5′-CGCAAATGGGCGGTAGGCGTGGCCATAACCCAATACCAAACG-3′ | ||
RT primer sequence: CO2 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGTAAAGGATGCGTAGGGATGG-3′ | ||
RT primer sequence: CO2 Light | 5′-CGCAAATGGGCGGTAGGCGTGCTAGTCCTGTATGCCCTTTTCC-3′ | ||
RT primer sequence: CO3 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGCTCCTGATGCGAGTAATACGG-3′ | ||
RT primer sequence: CO3 Light | 5′-CGCAAATGGGCGGTAGGCGTGCCTTTTACCACTCCAGCCTAG-3′ | ||
RT primer sequence: CYTB Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGGATAGTAATAGGGCAAGGACG-3′ | ||
RT primer sequence: CYTB Light | 5′-CGCAAATGGGCGGTAGGCGTGCAATTATACCCTAGCCAACCCC-3′ | ||
RT primer sequence: GAPDH | 5′-CGCAAATGGGCGGTAGGCGTGTGAGCGATGTGGCTCGGCT-3′ | ||
RT primer sequence: ND1 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGTTGTGATAAGGGTGGAGAGG-3′ | ||
RT primer sequence: ND1 Light | 5′-CGCAAATGGGCGGTAGGCGTGTCAAACTCAAACTACGCCCTG-3′ | ||
RT primer sequence: ND4 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGTGTTTGTCGTAGGCAGATGG-3′ | ||
RT primer sequence: ND4 Light | 5′-CGCAAATGGGCGGTAGGCGTGCCTCACACTCATTCTCAACCC-3′ | ||
RT primer sequence: ND5 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGTTTGGGTTGAGGTGATGATG-3′ | ||
RT primer sequence: ND5 Light | 5′-CGCAAATGGGCGGTAGGCGTGCATTGTCGCATCCACCTTTA-3′ | ||
RT primer sequence: ND6 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGGTTGAGGTCTTGGTGAGTG-3′ | ||
RT primer sequence: ND6 Light | 5′-CGCAAATGGGCGGTAGGCGTGCCCATAATCATACAAAGCCCC-3′ | ||
Siliconized polypropylene 1.5 mL G-tube | Bio Plas | 4167SLS50 | |
Sodium dedecyl sulfate | Biosesang | S1010 | |
Sodium deoxycholate | Sigma-Aldrich | D6750 | |
SUPERase In Rnase inhibitor | Thermo Fisher Scientific | AM2694 | |
SuperScript III reverse transcriptase | Thermo Fisher Scientific | 18080093 | Reverse transcriptase for library preparation |
SuperScript IV reverse transcriptase | Thermo Fisher Scientific | 18090010 | Reverse transcriptase for qRT-PCR |
SYBR gold nucleic acid gl stain | Thermo Fisher Scientific | S11494 | |
T4 polynucleotide kinase | New England Biolabs | M0201S | |
T4 RNA ligase 1 (ssRNA Ligase) | New England Biolabs | M0204 | |
T4 RNA ligase 2, truncated KQ | New England Biolabs | M0373 | |
Thermomixer | Eppendorf ThermoMixer C with ThermoTop | ||
Thymidine | Sigma-Aldrich | T9250 | |
Tris-borate-EDTA buffer (TBE) | TaKara | T9122 | |
Triton X-100 | Promega | H5142 | |
Ultralink Protein A sepharose beads | Thermo Fisher Scientific | 22810 | Protein A beads |
Ultrasonicator | Bioruptor | ||
Urea | Bio-basic | UB0148 | |
Vortex mixer | DAIHAN Scientific | VM-10 | |
Xylene cyanol | Sigma-Aldrich | X4126 | |
γ-32P-ATP (10 μCi/μL, 3.3 μM) | PerkinElmer | BLU502A100UC |