GSM4403852: Npm1c.HSPC_pCHiC_rep2; Mus musculus; OTHER
20-25 million cells were crosslinked by 1% formaldehyde for 10 minutes at room temperature and was then quenched by glycine. Cells were lysed with the help of homogenization. Chromatin digestion was achieved overnight using HindIII. Digested DNA ends were biotinylated by filling with biotin-14-dATP (Invitrogen) and underwent in-nucleus ligation using T4 DNA ligase at 16 °C overnight. Crosslink was then reversed by incubation with Proteinase K at 65  ̊C overnight. RNA was removed by adding RNase A and incubating at 37  ̊C for 1 hour. DNA was purified twice with Phenol:Chloroform:Isoamyl Alcohol. Extra Biotin-14-dATP was first removed using T4 DNA polymerase and incubating at 20  ̊C for 4 hours. 40 μg DDNA was sheared using Covaris S2 to achieve a target peak around 400 bp. Sheared DNA was subject to end repair and purification, followed by addition of dATP as A-tail. DNA fragments were size selected to enrich at 200-650 bp by double-sided SPRI beads (Invitrogen). Biotin-labeled fragments were pulled down using Dynabeads MyOne Streptavidin C1 (Invitrogen) and ligated to pre-annealed paired-end (PE) adapter on beads. Eventually, HiC DNA was amplified using PE PCR primers for 7-9 cycles. HiC library was purified twice using 1.8x SPRI beads. 500 ng of HiC library was used for hybridization with biotinylated RNA bait library (Agilent Technologies), which contained 39684 biotinylated RNA probes covering 22047 promoters. Hybridization was performed as per manufacture's protocol. Dynabeads MyOne Streptavidin T1 beads were utilized to pull down biotin-labeled DNA-RNA hybrids. Final PCR amplification and DNA purification of promoter capture HiC library were performed similarly as for HiC library, with the difference for 4 cycles of PCR. pCHiC were performed in biological duplicates of each cellular states.
GEO
GSM4403852