title: GSM3146552: MB_WT_GRO_rep2; Mus musculus; OTHER
library_construction_protocol: GRO-seq experiments were carried out as previously described (Core et al., 2008; Mahat et al., 2016). Five million cells (WT or MyoD KO C2C12 cells at MB and MT stages) were resuspended in cold swelling buffer (10 mM Tris-HCl pH7.5, 2 mM MgCl2, 3 mM CaCl2) on ice for 5 minutes and then lysed in lysis buffer (swelling buffer + 0.5% IGEPAL + 10% glycerol + 2 units/ml SUPERase In, and cOmplete protease inhibitors) followed by gently pipetting up and down for 20 times. After centrifuge, nuclei were sequentially washed with lysis buffer and freezing buffer (50 mM Tris-HCl pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA) and finally resuspended in 100 µl of freezing buffer and stored at -80 °C. Nuclear run-on (NRO) assays were performed with biotin-11-UTP as previously reported. In brief, 2× NRO master mix (10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 1% Sarkosyl, 250 µM ATP, GTP, CTP, 50 µM biotin-11-UTP, and 0.8 u/µl SUPERase In) was pre-equilibrated at 37 °C for 10 minutes. Then, 5×106 cells / 100 µl nuclei were added to the same volume 100 µl of 2×NRO master mix and incubated at 37 °C for 5 minutes. The run-on RNA (NRO-RNA) was extracted with 2 ml of TRIzol reagent (Invitogen) following the manufacturer's instructions. The NRO-RNAs were then fragmented with 5 μl of ice-cold 1 N NaOH for 10 minutes on ice. The reaction was neutralized by mixing with 25 μl of 1 M Tris-HCl pH 6.8 and fragmented biotinylated RNA was purified through incubation with 30 µl of Dynabeads™ M-280 Streptavidin beads (Thermo Fisher Scientific) in binding buffer (10 mM Tris-HCl pH 7.4, 300 mM NaCl and 0.1% Triton X-100) for 20 minutes at room temperature while rotating. Then, the beads were sequentially washed two times with high-salt wash buffer (50 mM Tris-HCl pH 7.4, 2 M NaCl and 0.5% Triton X-100), two times with binding buffer, and one timer with low-salt wash buffer (5 mM Tris-HCl pH 7.4, 0.1% Triton X-100). After elution from beads, biotinylated RNA was subject to 3' RNA adaptor ligation and incubated in a 10 µl reaction volume containing 50 pmol of 3' RNA adaptor (5'p-rGrArUrCrGrUrCrGrGrArCrUrGrUrArGrArArCrUrCrUrGrArArC-/3'InvdT/) (Integrated DNA Technologies, IDT), 10 nmol of ATP, 10 unites of T4 RNA ligase I (NEB), 40 unites SUPERase In and 10% PEG 8000 at 20 ̊C for 6 hours. Ligated RNA was enriched by Streptavidin beads and RNA extraction with TRIzol. The 5' ends of biotinylated RNA were then repaired with RNA 5´ Pyrophosphohydrolase (RppH) (NEB) and T4 polynucleotide kinase (PNK) (NEB) followed by Trizol extraction. Subsequently, the purified RNA was ligated to 5' RNA adaptor (5'-rCrCrUrUrGrGrCrArCrCrCrGrArGrArArUrUrCrCrA-3') (IDT) in a 10 µl reaction volume containing 50 pmol of 5' RNA adaptor, 10 nmol of ATP, 10 unites of T4 RNA ligase I (NEB), 40 unites SUPERase In and 10% PEG 8000 at 20 ̊C for 6 hours. Following the ligation, ligated RNA was purified by the third round Streptavidin bead enrichment and TRIzol extraction. The resultant RNA was reverse transcribed with RP1 primer (5'-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3') (IDT) using Superscript III RT enzyme (Thermo Fisher Scientific). A small portion of cDNA was serial diluted and subjected to test PCR amplification to determine optical PCR cycle. The full-scale PCR amplification was performed using Q5® High-Fidelity 2× Master Mix (NEB) with 12.5 pmol of RP1 primer and RPI-index primers (5' -CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3', in which 'NNNNNN' is an index sequence) (IDT). The optimal PCR cycle for full-scale amplification is 14. PCR products were PAGE separated and DNA from 140 bp to 350 bp was eluted from PAGE gel. The library was quantified and sequenced in the Illumina HiSeq 1500.
attributes.tag: GEO Accession
attributes.value: GSM3146552