title: GSM3212357: +dox_ssRNA-seq_rep1; Mus musculus; RNA-Seq
library_construction_protocol: [ATAC-seq extract and library construction protocol]   ATAC-seq was performed as described previously (1). Briefly, 500,000 cells were harvested and washed with 500 μl cold PBS and resuspended in 500 μl cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40), and nuclei were pelleted with low speed centrifugation (500g, 4°C, 10 min). Nuclei from 50,000 cells were resuspended in 25 μl TD Buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10% dimethlyformamide), and 2.5 μl Tn5 Transposase (Illumina, Cat #FC-121-130) and incubated at 37°C for 30 min. Fragmented DNA was purified using a MinElute kit (Qiagen, Cat#28206), and libraries were generated by PCR using NEB Next High-Fidelity 2x PCR Master Mix (NEB, Cat#M0541). An appropriate number of PCR cycles was determined by qPCR as described (2). Tagmentation was confirmed via Tapestation. Libraries were sequenced on HiSeq 2500 with paired-end reads (51bp). [ssRNA-seq extract and library construction protocol]  TRIzol (Thermo Fisher, Cat# 15596018) was used to isolate total RNA according to the manufacturer's instructions. For RNA-seq, genomic DNA was removed from total RNA using the TURBO DNA-free™ Kit (Thermo Fisher, Cat#AM1907), the quality of RNA was assessed using Agilent High Sensitivity RNA ScreenTape (Agilent, Cat#5067-5579), 2 μg of total RNA (with RIN® > 8) was depleted of ribosomal RNA using a Ribo-Zero Gold rRNA Removal Kit (H/M/R) (Illumina, Cat# MRZG126) according to manufacturer's instructions, and RNA was purified using RNA Clean & Concentrator-5 kit (Zymo Research, Cat#R1015). Purified mRNA was fragmented using NEBNext® Magnesium RNA Fragmentation Module (NEB, Cat#E6150S1) at 94°C for 5 min followed by purification and elution into 17.5 μl H2O. Dephosphorylation of the 3'phosphate group was performed with shrimp alkaline phosphatase (SAP) (Worthington, Cat#M0371S), followed by inactivation of phosphatase. Phosphorylation of RNA was performed with T4 Kinase (NEB, Cat# M0201S). Strand-specific cDNA libraries were prepared using NEBNext® Multiplex Small RNA Library Prep kit for Illumina (NEB, Cat#E7300S) according to manufacturer's instructions. Libraries were quantified using Qubit dsDNA HS assay kit (Thermo Fisher, Cat#Q32854), and the quality and average size of the libraries were assessed using Agilent D1000 ScreenTape (Agilent, Cat#5067-5582) on the Agilent 2200 Tapestation. Paired-end sequencing was performed using NextSeq 500/550 High Output v2 kit (75 cycles) (Illumina, Cat# FC-404-2005) on NextSeq 550 System. [ChIP-seq extract and library construction protocol]  ChIP and ChIP-seq libraries were prepared as described (Asp, P., Blum, R., Vethantham, V., Parisi, F., Micsinai, M., Cheng, J., . . . Dynlacht, B. D. (2011). Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci U S A, 108(22), E149-158. doi:10.1073/pnas.1102223108) and sequenced (51 bp, single-end reads) using an Illumina HiSeq2500 machine or (37bp, paired end) using an Illumina NextSeq 500 machine [MNase-seq extract and library construction protocol]  MNase-seq libraries were prepared as described (Asp, P., Blum, R., Vethantham, V., Parisi, F., Micsinai, M., Cheng, J., . . . Dynlacht, B. D. (2011). Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci U S A, 108(22), E149-158. doi:10.1073/pnas.1102223108) and sequenced (51 bp, single-end reads) using an Illumina HiSeq2500 machine or (37bp, paired end) using an Illumina NextSeq 500 machine [extract nuclei for Pro-seq ]  Nuclei were isolated from C2C12 cell as described (Core et al., 2008) with some modifications. Briefly, 3-4 15 cm2 plates of C2C12 cells (20 x106 cells) were washed twice with ice cold PBS and collected into 50 ml centrifuge tubes using a plastic cell scraper in cold room. Cells were resuspended in 5 ml of ice cold swelling buffer (20 mM Tris-HCl pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 1:10000 dilution of 20 U/μL SUPERase• In™ RNase Inhibitor) and allowed to swell on ice for 5 min. Cells were pelleted at 4 °C for 10 min (2000 rpm), cells were resuspended in 5 ml of lysis buffer tNuclei were isolated from C2C12 cell as described (5) with some modifications. Briefly, 3-4 15 cm2 plates of C2C12 cells (~ 20 x106 cells) were washed twice with ice cold PBS and collected using a cell scraper. Cells were resuspended in 5 ml of ice cold swelling buffer (20 mM Tris-HCl pH 7.5, 2 mM MgCl2, 3 mM CaCl2, 1:10000 dilution of 20 U/μL SUPERase In™ RNase Inhibitor) and allowed to swell on ice for 5 min. Cells were pelleted at 4 °C for 10 min (2000 rpm) and resuspended in 5 ml of lysis buffer twice (swelling buffer + 0.5 % NP40, + 10 % glycerol + 1:10000 dilution of 20 U/μL SUPERase  In™ RNase Inhibitor) and gently pipetted up and down 20 times using a p1000 tip with the end cut off to reduce shearing, the nuclei were pelleted and resuspended in 1ml Freezing buffer (50 mM Tris-HCl pH 8.3, 40 % glycerol, 5 mM MgCl2, 0.1 mM EDTA), and transferred to a 1.5 ml centrifuge tube. Nuclei were spun down and resuspended in Freezing Buffer (5x106 nuclei per 100 μl). wice (swelling buffer + 0.5 % NP40, + 10 % glycerol + 1:10000 dilution of 20 U/μL SUPERase• In™ RNase Inhibitor) and gently pipetted up and down 20 times using a p1000 tip with the end cut off to reduce shearing, the nuclei were pelleted and resuspended in 1ml Freezing buffer (50 mM Tris-HCl pH 8.3, 40 % glycerol, 5 mM MgCl2, 0.1 mM EDTA), and transferred to a 1.5 ml centrifuge tube. Nuclei were spun down and resuspended in Freezing Buffer (5x106 nuclei per 100 μl). [Pro-seq library construction protocol]  Proseq was based on a published Proseq protocol (6) with some modifications. Nuclear run-on reactions were performed by adding 100 μl of nuclei (5x106) to 100 μl of preheated (37°C) 2x nuclear run-on (NRO) master mix (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 300 mM KCl , 1 mM DTT, 250 mM ATP, 250 mM GTP, 50 mM biotin-11-CTP, 50 mM biotin-11-UTP, and 1% Sarkosyl), pipetting the mixture 15 times, and incubating reactions at 37°C for 5 min. Reactions were stopped with Trizol LS, and RNA was extracted once with chloroform and precipitated with ethanol (vol/vol), and the pellet was washed in 75% ethanol once and re-dissolved in DEPC-H2O. Next, RNA fragmentation was performed using base hydrolysis, biotin RNA enrichment was performed using pre-washed streptavidin beads once at room temperature for 30 min, and the beads were washed twice in ice-cold high-salt wash buffer (50 mM Tris-HCl pH 7.4, 2 M NaCl and 0.5 % (vol/vol) Triton X-100 in DEPC H2O), twice in binding buffer (10 mM Tris-HCl pH 7.4, 300 mM NaCl and 0.1 % Triton X-100), and once in low-salt wash buffer (5 mM Tris-HCl pH 7.4 and 0.1 % Triton X-100). RNA was extracted from beads using Trizol, ethanol precipitated, and dissolved in H2O. The 5' cap of RNA was removed from unhydrolyzed short nascent RNA using 5U RNA 5' pyrophosphohydrolase (RppH), and the 5' OH generated by base hydrolysis was then converted to 5' phosphate by treatment with 25U T4 polynucleotide kinase (PNK). RNA was extracted with Trizol and dissolved in H2O, and libraries were prepared using NEB Next Multiple Small RNA library Prep set for Illumina (NEB, Cat#E7300S), following manufacturer's indications. Libraries were sequenced on the Illumina NextSeq 550.