title: GSM9042977: RNAseq, MuSCs, Control, replicate 1; Mus musculus; RNA-Seq
library_construction_protocol: For RNA-seq: IMR90 or IMR90/MyoD cells total RNA was extracted using Quick-RNA Microprep KIT (Zymo Research, R1051) following the manufacturer's recommendation. RNA concentration was measured on Qubit (Invitrogen). For ATAC-seq: IMR90 or IMR90/MyoD cells were freshly collected directly processed for the library preparation. For ChIP-seq: IMR90 or IMR90/MyoD cells were fixed with 1% FA for 10 minutes at RT followed by quenching with glycine (final concentration 200 mM) for 15 minutes on ice. Nuclei were then extracted in hypotonic buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.05% NP-40, 1 mM PMSF and 1x protease inhibitor) and lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, pH 8.0, 0.5% SDS, 0.5% NP-40, 1 mM PMSF and 1x protease inhibitor and lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, pH 8.0, 0.5% SDS, 0.5% NP-40, 1 mM PMSF and 1x protease inhibitor). Chromatin was sheared with sonicator (S2 Covaris) to an average DNA fragment length of 200-500bp. 15µg of sonicated chromatin were diluted in 500µl RIPA Dilution Buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, pH 8.0, 0.1% Sodium Deoxycholate, 0.7% NP-40, 1 mM PMSF and 1x protease inhibitor), and precleared for 3 hours at 4°C with Protein A/G dinabeads. In parallel, 10µl of CTCF antibody (CST Cat#3418S), or 1µg of rabbit IgG (Santa Cruz #sc-2027) were prebound to Protein A/G dynabeads in 500µl of PBS containing 5 µg/ml BSA, for 3 hours at 4°C. Beads bound antibody were mixed with precleared chromatin and incubated overnight at 4°C on a rotator. Chromatin bound fraction was washed with RIPA washing buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, pH 8.0, 0.5% Sodium Deoxycholate, 1% NP-40) for 4 times, followed by LiCl washing buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, pH 8.0, 1% Sodium Deoxycholate, 1% NP-40), and TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), each wash for 10 minutes at 4°C. Chromatin was then eluted in elution buffer (1% SDS in TE buffer) at 65°C for 6 hours 600 RPM rotation. For CUT&RUN: IMR90 or IMR90/MyoD cells or Muscle Stem Cells (MuSCs) were used as input. CUT&RUN experiments were performed using the CUT&RUN Assay Kit (CST #86652) with the following modification: cells were briefly fixed with 0.1% FA for 2 minutes at RT followed by quenching with glycine; antibody binding was performed O/N at 4C. Antibodies used: anti-MYOD (BD Bioscience, #554130), anti-H4K5/K12AcMe, anti-panAcMe, anti-H3K27ac (Active Motif #91193), anti-H3K4me3 (CST #9751), anti-H3K4me1 (Active Motif #39635), anti-H3K27me3 (Active Motif #39155), anti-H3K9me3 (Active Motif #39161). For HiChIP: IMR90 or IMR90/MyoD cells were fixed with 2%FA for 10 minutes at RT followed by quenching with STOP solution1 according to ARIMA HiC+ protocol. Digestion was performed overnight at 37C. Biotinylated enriched fragments were pulled down with anti-H3K27ac (Active Motif #91193) or anti-CTCF (Active Motif #91285). Experiments were performed using the ARIMA HiC+ kit according to manufacturer's protocol. For RNA-seq: Equal inputs of total RNA (10-100 ng) were used to generate stranded total RNA libraries for sequencing using the Illumina® Stranded Total RNA Prep, Ligation with Ribo-Zero Plus (Illumina #20040525). ERCC RNA Spike-in mix was added at the start of the protocol according to manufacturer's instruction (Thermo Fisher Scientific #4456740). Libraries were sequenced at a depth of ~50M per library on NOVASeqS4 PE 2x100. For ATAC-seq: Freshly collected cells were subjected to ATAC-seq library preparation using the ATAC-seq Kit (Active Motif #53150) according to manufacturer's protocol. For ChIP-seq: Precipitated chromatin and 10ng of input were used for library preparation using Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences #21096) according to manufacturer's protocol. Obtained libraries were sequenced at a depth of ~100M per library on NOVASeqS4 PE 2x100. For CUT&RUN: Libraries have been generated using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB #E7645S) according to manufacturer's protocol. Libraries were sequenced at a depth of ~20M per library on NOVASeqS4 PE 2x100. For HiChIP: H3K27ac or CTCF enriched material was then used as input for library preparation using Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences #21096) according to ARIMA HiChIP library preparation protocol. Obtained libraries were sequenced at a depth of ~300M per library on NOVASeqS4 PE 2x100.