GSM2948285: Hi-C preB young 1; Mus musculus; Hi-C
RNA-seq: For nuclear RNA-seq nuclei were isolated from 1-10 x 106 flow sorted B lymphocytes by incubation in 50 mM Tris-HCl pH 7.5, 140 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 0.4% NP40 for 5 min on ice followed by centrifugation at 500 x g. Total and nuclear RNA was isolated with a RNeasy mini kit (Qiagen) and treated with Turbo DNAse (Ambion).  Total RNA was depleted of ribosomal RNA using a Ribo-Zero Gold rRNA removal kit (Human/Mouse/Rat:Illumina) according to the manufacturer's instructions.  miRNA-seq: Short RNAs were isolated from 1-10 x 106 flow sorted B lymphocytes using a mirVANA kit (Ambion/ThermoFisher).  ATAC-seq: ATAC-seq was performed directly on 5 x 104 flow sorted B lymphocytes.  ChIP-seq: Flow sorted B lymphocytes were fixed in 1% formaldehyde for 10 min at RT, quenched with 125mM glycine, washed with PBS and pellets snap frozen for storage at -80°C. Cells were lysed (150mM NaCl/25mM Tris pH 7.4/5mM EDTA/0.1% Triton/1% SDS/protease inhibitors; + 20mM Sodium Butyrate for H3K27ac), chromatin sonicated and diluted 5-10 times (0.01% SDS/1.1% Triton/1.2mM EDTA/16.7mM Tris pH 8.1/167mM NaCl; or for H3K27ac in RIPA buffer: 10 mM Tris-HCl pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate) and incubated with antibody overnight at 4°C before addition of protA-G magnetic beads. For H3K27ac, antibody was instead pre-incubated with beads and washed with RIPA buffer before incubation with chromatin at 4°C overnight. Beads were washed once with Low Salt buffer (0.1% SDS, 0.1% SDS, 2mM EDTA, 20mM Tris pH 8.0, 150mM NaCl), twice with High Salt buffer (0.1% SDS, 0.1% SDS, 2mM EDTA, 20mM Tris pH 8.0, 500mM NaCl) and twice with TE (10mM Tris pH 8.0, 1mM EDTA). For CTCF the last High Salt buffer was substituted with LiCl Buffer (0.25M LiCl, 1% IGEPAL, 1% deoxycholic acid, 1mM EDTA, 10mM Tris, pH 8.1). For H3K27ac the first 3 washes were performed with RIPA buffer. DNA was eluted in 0.1M NaHCO3/1% SDS and reverse-crosslinked with Proteinase K (Ambion); ChIP and input DNA was purified using the QIAquick PCR Purification Kit (Qiagen).  Hi-C/Promoter Capture Hi-C: Hi-C and Promoter CHi-C libraries were generated as described (Schoenfelder et al., 2015), with modifications as described below. 3.2-3.5 x 107 pre-B cells, pooled from several FACS sorts, were used for each library. DNA digestion was performed with 1500 units HindIII (NEB) per 5 million cells. After the biotin fill-in, in-nucleus ligation was performed as described previously (Nagano et al., 2015) for four hours at 16°C (5.5 ml 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 µg/ml BSA + 50 units T4 DNA ligase (Life Technologies)). An additional Proteinase K incubation (65 µl of 10mg/ml) for two hours was included after the overnight incubation, preceding RNase A treatment, two sequential phenol/chloroform (Sigma) extractions and DNA precipitation including three 70% ethanol washes. Biotin removal from non-ligated fragment ends was followed by phenol/chloroform purification and DNA precipitation. RNA-seq: Paired-end strand-specific libraries were generated as described (Parkhomchuk et al., 2009) except polyA+ RNA selection was omitted, first strand cDNA synthesis was performed with random hexamer primers, and double-stranded cDNA was fragmented with a Covaris E220 sonicator.   miRNA-seq: Libraries were generated using the NEBNext small RNA library prep set for Illumina with 15 cycles of PCR. Transfer RNA and ribosomal RNA derived fragments were removed from the libraries by 'double-sided' AMPure XP size selection as described in the user manual.  ATAC-seq: Atac-seq was performed as described in Buenrostro et al. (2015).  ChIP-seq: Library preparation was performed from 0.2-0.8 ng of purified DNA using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® with the following modifications: Illumina Tru-Seq adaptors were used and library amplification was performed with the KAPA PCR Amplification kit (KAPA, Cat. KK2501) using 15 cycles.   Hi-C/Promoter Capture Hi-C: Libraries were generated essentially as described in Schoenfelder et al. (2015).
GEO Accession
GSM2948285