GSM4307233: RACE-Rep1-WT-TCRD; Mus musculus; RNA-Seq
For Repertoire-seq, a mixture 107 cell equivalents of RNA and 1 μM oligo(dT) primer in 8μl nuclease-free water was heated to 65°C for 5 min and cooled down on ice for at least five minutes to snap-anneal the oligo(dT) primer. The reaction was then adjusted to 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, 0.5 mM dNTPs and 5 mM dithiothreitol, before addition of 2μl Superscript II (ThermoFisher), add 1μl 25uM 5′RACE adapter sequence (5′-GTCGCACGGTCCATCGCAGCAGTCACArGrGrG-3′) and 1μl in a final volume of 20 μl. The reaction was incubated for two hours at 50°C to synthesize cDNA and add RACE adapter by template switching. Reverse transcriptase was then inactivated by incubation at 85°C for 2 minutes. For ChIP-seq,  CD3-stimulated  thymus was removed,cells were filtered through nylon mech. Red cells were lysed in AcK buffer.Cells were perforated and dealed with MNase, chromatin were isolated by immunoprecipitation. For Hi-C, libraries were prepared using in-situ HiC protocol described in (Rao et al., 2014) with small modification. For 4C, 3C sample preparation followed a previously described protocol(Hagège et al., 2007) with small modification, 3C samples were digested overnight at 37°C with 10 U of NlaIII.After purification,ligation and PCR amplification,the library preparation followed the protocol described in ChIP libary construction. For ChIP-seq, ChIP product through end repair, dA-tailing and linker ligation, barcodes and Illumina adapters were then added by PCR amplification. The libraries were purified with QiaQuick PCR purification reagents (Qiagen) and size selection by 0.7× and 0.2×Ampure XP beads (Beckman, A63880). For Repertoire-seq, the library preparation followed the protocol described in ChIP libary preperation after PCR amplification. For 4C, the library preparation followed the protocol described in ChIP libary construction after PCR amplification. Hi-C libraries were prepared using in-situ HiC protocol described in (Rao et al., 2014) with small modification.