Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

1. Preparing the shRNA Plasmid DNA from Escherichia coli

1. Generation of clonal bacterial colonies carrying the shRNA plasmids
   1. Obtain glycerol stocks of E. coli carrying target-specific shRNA plasmids and a control plasmid from commercial sources (Table of Materials).
      NOTE: Three different shRNA plasmids were used, targeting different regions of the murine Adamtsl2 mRNA. One shRNA was selected to target the 3'-untranslated region (3'UTR) of Adamtsl2 to facilitate rescue experiments with expression plasmids encoding recombinant full-length ADAMTSL2 or individual ADAMTSL2 protein domains. In addition, a scrambled shRNA plasmid was included as a negative control. Details of the shRNA sequences are provided in Figure 1A.
   2. Thaw the shRNA bacterial glycerol stock at room temperature (RT). Transfer 10 µL of bacterial glycerol stock onto a Luria-Bertani (LB) agar plate supplemented with 100 µg/mL ampicillin (LB-Amp).
      NOTE: LB-Amp plates are prepared by autoclaving 1 L of LB medium (for 1 L: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, adjust pH to 7.0) together with 12 g of agar. Let the medium cool down to ~50 °C and add ampicillin (stock solution: 50 mg/mL in sterile water) to a final concentration of µg/mL (LB-Amp agar). Immediately pour ~20 mL of LB-Amp agar in a 10 cm Petri dish and let the agar solidify before use. 1 L of LB-Amp agar is typically sufficient to pour about 40 10 cm Petri dishes. Petri dishes containing LB-Amp agar are stable for at least one month if stored sealed at 4 °C in the dark.
   3. Spread bacteria with sterile Drygalski spatula or any other appropriate method to achieve single bacterial colonies. Incubate bacterial plates upside down overnight at 37 °C.
2. Plasmid preparation
   1. The next morning, remove the Petri dish with the individual bacterial colonies and store at 4 °C to avoid overgrowth of the bacterial colonies and the formation of satellite colonies. Seal the Petri dish if stored overnight or longer.
   2. In the afternoon, add 5 mL of LB-Amp medium into a polypropylene bacterial culture tube. Select a single bacterial colony from the Petri dish cultured overnight with a pipette tip and inoculate LB-Amp medium by ejecting the pipette tip in the bacterial culture tube containing the LB-Amp medium.
   3. Incubate bacterial culture overnight in a shaker at 250 rpm at 37 °C with the lid loosely attached to allow for aeration.
   4. Take out bacterial culture tube the next morning and keep at 4 °C to avoid bacterial overgrowth. In the afternoon, resuspend bacteria by vortexing and transfer 1 mL of the overnight bacterial culture in 50 mL of LB-Amp medium in a 250 mL conical flask. Incubate overnight in a shaker at 250 rpm at 37 °C.
   5. On the next morning, transfer bacteria to a 50 mL disposable centrifuge tube and centrifuge bacteria at 6,000 x g at RT for 15 min. Remove the medium and continue with step 1.2.6.
      NOTE: If plasmid DNA cannot be isolated immediately, remove the LB-Amp medium after the centrifugation step and store the bacteria pellet at -20 °C.
   6. Follow instructions for plasmid preparation kit (midi scale) to extract the plasmid DNA from the bacteria. Assess plasmid DNA quality by measuring the A₂₆₀/A₂₈₀ ratio using a spectrophotometer.
      NOTE: An A₂₆₀/A₂₈₀ ratio of >1.8 is desirable, indicating high purity of the plasmid DNA preparation. A lower A₂₆₀/A₂₈₀ ratio suggests contamination with protein or insufficient removal of extraction reagents. Additional plasmid purification steps may be required.

2. Culturing and Transfection of C2C12 Cells and Puromycin Selection

1. C2C12 cell culture
   1. Thaw C2C12 cells (Table of Materials) quickly in a 37 °C water bath and pour cells in sterile disposable 15 mL centrifuge tube containing 8 mL of Dulbecco's modified Eagle medium (DMEM) medium supplemented with 100 units of penicillin and streptomycin antibiotics (serum-free DMEM) in a cell culture hood. Centrifuge cells for 3 min at 160 x g at RT.
   2. Aspirate supernatant and resuspend the cell pellet in 10 mL of serum-free DMEM supplemented with 10% fetal bovine serum (FBS) (complete DMEM). Transfer cells to 10 cm tissue culture treated plastic dishes. Incubate cells in a humidified incubator at 37 °C in a 5% CO₂ atmosphere.
   3. On the next day, replace the medium with fresh complete DMEM.
   4. Expand low passage C2C12 cells on 3−4 10 cm dishes. When the C2C12 cells reach 50−60% confluence, aspirate the medium and rinse C2C12 cells with 10 mL of phosphate-buffered saline (PBS).
   5. Add 1 mL of 0.25% trypsin-EDTA and incubate for 2 min at RT. Monitor cell detachment under a microscope and extend the incubation time if necessary, until most of the cells are detached.
      NOTE: Carefully tapping the dish can help to dislodge the cells.
   6. When most of the cells are detached, add 10 mL of complete DMEM to the dish, pipette the volume up and down several times, and transfer cell suspension to a sterile disposable 15 mL centrifuge tube. Centrifuge cells for 3 min at 160 x g at RT. Aspirate supernatant and resuspend the cell pellet in 2 mL of freezing medium (10% dimethyl sulfoxide [DMSO]/90% FBS).
   7. Transfer two 1 mL aliquots per 10 cm dish in cryovials. Incubate in a cell-freezing container filled with RT isopropanol for at least 24 h in a -80 °C freezer resulting in a freezing rate of about -1 °C per min to avoid the formation of ice crystals. Store cells for long-term use in the vapor phase of liquid nitrogen.
      NOTE: The vendor recommends using C2C12 cells up to passage number 15. Authors' experiences also showed that lower passage number cells showed more consistent and rapid differentiation into myotubes. It is essential to maintain C2C12 cells at low cell density (<50% confluence) to avoid premature onset of differentiation. Differentiating or differentiated C2C12 cells cannot be reverted into the original myoblast state and must be discarded.
2. Preparing C2C12 cells for transfection
   1. Culture undifferentiated C2C12 cells in a 10 cm dish until they reach 50−60% confluence. Aspirate the medium, rinse C2C12 cells with 10 mL of PBS, and add 1 mL of 0.25% trypsin-EDTA. Incubate for 2 min at RT, monitor cell detachment under a microscope, and extend the incubation time if necessary, until most of the cells are detached.
      NOTE: Carefully tapping the dish can help to dislodge the cells.
   2. When most of the cells are detached, add 10 mL of complete DMEM to the dish and transfer cell suspension to a sterile disposable 15 mL centrifuge tube. Centrifuge cells for 3 min at 160 x g at RT. Aspirate supernatant and resuspend the cell pellet in 4 mL of complete DMEM.
   3. Combine 10 µL of cell suspension with 10 µL of trypan blue in a 1.5 mL reaction tube, mix by pipetting up and down and carefully flicking the reaction tube, and transfer cells to a counting slide. Determine the cell number/mL using an automated cell counter.
   4. Dilute C2C12 cells to 50,000 cells/mL and seed 100,000 cells (2 mL) per well in a 6 well plate to achieve about 40−50% confluence after overnight incubation. Incubate cells in a humidified incubator at 37 °C in a 5% CO₂ atmosphere.
3. Transfection of C2C12 cells with the shRNA plasmid using polyethylenimine (PEI) and puromycin selection
   1. Preparation of PEI stock solution
      1. Dissolve 16 mg of PEI in 50 mL of sterile distilled water (concentration of stock solution: 0.32 mg/mL) in a 50 mL glass media bottle with a screw cap. Incubate the solution at 65 °C for 1 h and vortex the solution vigorously several times during the incubation period to completely dissolve the PEI.
      2. Adjust to pH 8 by adding 15 µL of 1N HCl per 50 mL.
         CAUTION: HCl is corrosive. Concentrated HCl should be handled under the fume hood. Investigators should wear appropriate personal protective equipment.
      3. Freeze the PEI solution at -80 °C for 1 h with a loosely attached lid and rapidly thaw at 37 °C in a water bath to further enhance solubility. Repeat the freeze-thaw cycle 3x.
      4. Store PEI stock solution in 0.5 mL aliquots for one-time use at -20 °C.
   2. Transfection of C2C12 cells
      1. Combine 25.5 µL (8.5 µL/µg plasmid DNA) of the PEI stock solution with 100 µL of 25 mM NaCl in a 1.5 mL reaction tube and incubate for 5 min at 37 °C. Combine 3 µg of the plasmid DNA with 100 µL of 25 mM NaCl and incubate for 5 min at 37 °C.
      2. Combine the entire volume of diluted PEI reagent with the diluted plasmid and mix by gently pipetting up and down. Incubate for 25 min at 37 °C.
      3. During the incubation time, change the medium of the C2C12 cells intended for transfection to DMEM without FBS or antibiotics.
      4. Add the PEI/DNA transfection mix to C2C12 cells drop by drop and mix constantly by carefully moving the cell culture dish. Incubate in a humidified incubator at 37 °C in a 5% CO₂ atmosphere. After 6 h, change the medium to complete DMEM.
   3. Puromycin selection
      1. 24 h after transfection, switch medium to selection medium (complete DMEM plus 5 µg/mL puromycin). Continue puromycin selection until puromycin-resistant C2C12 cells are obtained, which typically takes 10−14 days.
      2. Expand puromycin resistant C2C12 cells at low cell density (<50−60% confluence), i.e., to maintain the undifferentiated state and cryopreserve of 6−10 vials for future experiments as described in steps 2.1.4−2.1.7.
         NOTE: Maintain puromycin-resistant C2C12 cells in the presence of 5 µg/mL puromycin for routine cell culture and expansion. However, puromycin was omitted in the differentiation experiments.

3. Phenotypic Analysis of C2C12 Differentiation

NOTE: The methods described below can easily be adapted for general phenotypic analysis of C2C12 myoblast differentiation into myotubes by varying the specific antibodies used in Western blotting or the gene specific primers used in the quantitative polymerase chain reaction (qPCR) analysis.

1. C2C12 differentiation protocol and brightfield microscopy
   1. Seed 150,000 cells/well in a 12 well plate. Culture puromycin-resistant C2C12 stable cells in a 12 well plate in selection medium in a humidified incubator at 37 °C in a 5% CO₂ atmosphere. Culture C2C12 cells in complete DMEM until they reach ~95% confluence.
   2. To induce differentiation of C2C12 cells, replace complete DMEM with serum-free DMEM (day 0 of differentiation). Change medium every two days and follow C2C12 myotube formation using an inverted bright field microscope with camera.
      NOTE: Many protocols use 2% horse serum to differentiate C2C12 cells into myotubes. In authors' hands, removing the serum entirely had a similar effect. Insulin is described as an additive to the cell culture medium to accelerate C2C12 differentiation. However, in the current protocol, C2C12 cells reliably differentiated into myotubes within 3−5 days after serum deprivation and addition of insulin was deemed unnecessary.
2. Myosin heavy chain (MyHC) immunostaining to visualize myotubes
   1. Seed 50,000 puromycin-resistant C2C12 cells per chamber in an 8 well chamber slide in 500 µL of complete DMEM. Culture cells until they reach 95% confluence in complete DMEM (about 24 h).
   2. To induce differentiation, switch from complete DMEM to 500 µL of serum-free DMEM (day 0 of differentiation). At the desired time point(s), aspirate the medium and rinse cells 3x with 0.5 mL of PBS.
      NOTE: Perform all following steps at RT.
   3. Fix cells with 0.2 mL of 4% paraformaldehyde (PFA) diluted in PBS for 15 min. Rinse cells with 0.5 mL of PBS 3x for 5 min each.
      CAUTION: PFA is hazardous. Take appropriate precautions and discard paraformaldehyde solution as hazardous waste.
   4. Quench PFA with 0.2 mL of 0.5 M glycine in PBS for 5 min. Rinse cells with 0.5 mL of PBS 3x for 5 min each.
   5. Incubate cells with 0.2 mL of 0.1% non-ionic surfactant/detergent (Table of Materials) in PBS for 10 min to permeabilize the cell membrane. Block with 0.2 mL of 5% bovine serum albumin in PBS for 1 h. Rinse cells with 0.5 mL of PBS 3x for 5 min each.
   6. Incubate cells with 0.2 mL of MyHC antibody (1:200 in PBS) for 2 h. Rinse cells with 0.5 mL of PBS 3x for 5 min each.
   7. Incubate cells with 0.2 mL of goat-anti mouse rhodamine red-conjugated secondary antibody (1:200 in PBS). Rinse cells with 0.5 mL of PBS 3x for 5 min each.
   8. After removing PBS quantitatively, add one drop of mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) per chamber. Coverslip and cure mounting medium according to the manufacturer's instructions. Seal with nail polish.
   9. Observe C2C12 cells using a fluorescence microscope using the appropriate filter set.
3. Assessing knockdown efficiency by quantitative real-time polymerase chain reaction (qRT-PCR)
   1. Culture C2C12 cells under differentiation conditions in 12 well plates as described in section 3.1.
   2. At the desired time point(s), remove the differentiation medium, rinse the cells once with 1 mL of PBS, and add 0.5 mL of RNA extraction reagent (Table of Materials) per well. Lyse the cells by carefully pipetting up and down and transfer cell lysate to a sterile 1.5 mL reaction tube. Isolate the RNA by carefully following the manufacturer's protocol.
      CAUTION: The RNA extraction reagent contains phenol. It should be used under a fume hood. Collect the RNA extraction reagent waste and dispose of as hazardous waste. Investigators should wear appropriate personal protective equipment.
   3. Dissolve the final RNA pellet in 20 µL of diethyl pyrocarbonate (DEPC)-treated water. Determine the quantity and quality of the RNA preparation using a spectrophotometer and determine the A₂₆₀/A₂₈₀ ratio as a quality measure.
      NOTE: A typical yield from 1 well of a 12 well plate is 5−7 µg of total RNA with an A₂₆₀/A₂₈₀ ratio of 1.8−2.
   4. To digest residual co-purified genomic DNA, dilute 1 µg of RNA in a total volume of 8 µL of DEPC-treated water in a PCR tube. Add 1 µL of 10x reaction buffer and 1 µL of DNase I (1 unit/µL) (Table of Materials) and incubate for 15 min at RT. Add 1 µL of stop solution (25 mM EDTA) and incubate at 70 °C for 10 min.
   5. To generate cDNA, combine 10 µL of DNase treated RNA with 10 µL of 2x reverse transcriptase master mix, containing the reverse transcriptase, random primers, 4 mM dNTP mix (1 mM each of dATP, dTTP, dGTP, and dCTP), and reverse transcriptase reaction buffer. Incubate the reaction in a thermocycler using the program as recommended by the manufacturer. Dilute cDNA with water in a 1:5 ratio.
   6. To prepare the qRT-PCR reaction, combine 5 µL of SYBR green qPCR master mix with 0.5 µL of gene-specific forward and reverse primers (stock: 10 µM), respectively, and 2 µL of DEPC-treated water per reaction. Pipette qPCR reaction in one well of a 96 well qRT-PCR plate and add 2 µL of diluted cDNA. Set up three technical replicates for each biological replicate and include a housekeeping gene such as Gapdh or Hprt1.
   7. Amplify the PCR product with a qRT-PCR thermocycler using the following program: 50 °C for 2 min (uracil-DNA glycosylase [UDG] activation), 95 °C for 2 min (dual-lock DNA polymerase activation), 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 1 min.
   8. Quantify qRT-PCR results using the DDCt method normalizing to a housekeeping gene, such as Gapdh or Hprt1.
4. Assessing knockdown efficiency by Western blotting
   1. Seed 300,000 puromycin-resistant C2C12 cells per well in a 6 well plate for Western blot analysis. After 24 h, change medium to serum-free DMEM to initiate differentiation (day 0 of differentiation).
   2. At the desired time point(s), collect two 1 mL of serum-free conditioned medium in a 1.5 mL reaction tube and centrifuge for 5 min at 500 x g at RT to remove detached cells and cell debris.
      NOTE: This step is necessary only if ECM proteins or other secreted proteins in conditioned medium are investigated. If the protein of interest is localized in the cytoplasm or is membrane bound, aspirate the cell culture medium and proceed with the cell lysis steps (3.4.7−3.4.12). Cell lysis should be performed in parallel to the protein precipitation from serum-free conditioned medium to minimize proteolytic degradation and other unintended consequences of prolonged storage of the cell layer.
   3. After centrifugation, transfer 1 mL of conditioned medium in a new 1.5 mL reaction tube and precipitate proteins by adding 0.391 mL of a mixture of trichloroacetic acid (TCA) and non-ionic surfactant/detergent. Briefly vortex the mixture and incubate for 10 min on ice.
      NOTE: Prepare the protein precipitation mixture directly before use by combining 0.252 mL of 55% TCA and 0.139 mL of 1% non-ionic surfactant/detergent per mL of conditioned medium and vortex briefly. The solution becomes turbid.
      CAUTION: TCA is corrosive and must be handled appropriately.
   4. Pellet the precipitated proteins at >16,000 x g for 10 min at 4 °C. Discard the supernatant.
      CAUTION: The supernatant contains TCA and should be collected separately and disposed of as hazardous material.
   5. Wash the protein pellet 3x with ice-cold acetone and centrifuge each time at >16,000 x g for 10 min at 4 °C.
   6. Air-dry the protein pellet for 3−4 min at RT and dissolve in 50 µL of 1x sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (50 mM Tris pH 6.8, 2% SDS, 6% glycerol, and 0.004% bromophenol blue, supplemented with 5% β-mercaptoethanol). Boil the sample for 5 min at 95 °C and use for Western blotting to detect the desired target protein using standard procedures.
      CAUTION: β-Mercaptoethanol is odorous and toxic and must be handled in a fume hood.
   7. For intracellular and membrane bound proteins, rinse the cell layer once with 2 mL of PBS.
   8. Add 1 mL of PBS and dislodge cells by scraping them of the well using a cell scraper. Collect cells in a 1.5 mL reaction tube and centrifuge at 3,420 x g for 3 min at 4 °C. Wash the cell pellet 3x with 1 mL of PBS and centrifuge at 3,420 x g for 3 min at 4 °C each time.
   9. To lyse the cells, resuspend the cell pellet in 0.2 mL of lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, and 1 mM sodium orthovanadate) supplemented with 1x EDTA-free protease inhibitor cocktail reagent and incubate for 30 min on ice.
   10. Ultrasonicate for 15 s on ice with a power output setting of 10 at an operating frequency of 23 kHz.
   11. Remove cell debris by centrifugation at 13,680 x g for 20 min at 4 °C. Collect supernatant in a new 1.5 mL reaction tube and determine the protein concentration using a commercial Bradford assay or any other suitable method.
   12. For each sample, combine 100 µg of protein with 5x SDS-PAGE sample buffer in a total volume of 60 µL and boil for 5 min at 95 °C. Analyze samples by standard Western blotting procedures for the presence of the desired target protein.