GSM6605833: RNA-seq_Old_rep3; Mus musculus; RNA-Seq
For the capture Hi-C libraries, whole genome Hi-C libraries were first generated as previously described. To enrich the IgH locus (mm10, chr12: 113,201,001 - 116,030,000), SureSelect Target Enrichment probes (Table S3) with 2× tiling density were designed and manufactured by Agilent (Agilent Technologies Inc.). Hi-C libraries were hybridized to probes as specified by the manufacturer. Enriched libraries were sequenced using Illumina NextSeq sequencer to generate paired-end 150-bp reads. Genome-wide in situ Hi-C was performed with Rag2-/- young and old primary pro-B cells, and Rag2-/-Ebf1+/+Pax5+/+, Rag2-/-Ebf1+/-Pax5+/+ and Rag2-/-Ebf1+/−Pax5+/− pro-B cell lines using the Arima Hi-C Kit (Arima Genomics), including KAPA Hyper Prep indexing and library amplification (catalog no. KK8500, Roche Molecular Systems Inc) according to the manufacturer's instructions. For each assay, 1X10^6 cells to be used as the input materials and two biological replicates were performed for each group. Samples were sequenced 2×150 bp on an Illumina NovaSeq instrument. For the ChIP-seq libraries, Young and old primary Rag2-/- pro-B cells were crosslinked with 1% formaldehyde (Sigma) for 10 min, quenched with 125 mM of glycine and lysed in lysis buffer containing 1% SDS. Chromatin was sheared by Bioruptor (Diagenode) and followed by immunoprecipitation with specific antibodies. For each immunoprecipitation assay, 1×10^6 cells was used. Antibody information anti-H3K27ac (Active Motif, # 39133; 1:500), anti-H3K27me3 (Diagonode, #C1541005; 1:500), anti-CTCF (Abcam, # ab70303; 1:250), anti-Rad21 (Abcam, # ab992; 1:500). Total RNA was prepared using the Direct-zol RNA Miniprep Kit (# R2051, Zymo Research) according to manufacturer's instructions. RNA libraries for Rag2-/-Ebf1+/+Pax5+/+, Rag2-/-Ebf1+/-Pax5+/+ and Rag2-/-Ebf1+/−Pax5+/− pro-B cell lines (two replicates for each group) were prepared using SMARTer Stranded Total RNA-Seq Kit v2 (# 634412, Takara Bio USA, Inc.) and sequenced 1×100 bp on an Illumina NovaSeq instrument. Total RNA was prepared using the Direct-zol RNA Miniprep Kit (# R2051, Zymo Research) according to manufacturer's instructions. The RNA libraries for young and old Rag2-/- primary pro-B cells (four biological replicates for each group) were prepared using the NEXTFLEX Rapid Directional RNA-Seq Kit (NOVA-5138-07, PerkinElmer) and sequenced 2×75 bp on an Illumina NovaSeq instrument.