GSM3955233
GSM3955233: H9_rep3_CNCC_V5_ChIP; Homo sapiens; ChIP-Seq
Approximately 7.5 million cells were fixed with 1% formaldehyde for 10 minutes, quenched with a final concentration of 0.125M glycine. Chromatin was sheared on a Covaris sonicator to approximately 500-2000bp fragments. Salt was added and chromatin was immunoprecipitated overnight at 4°C with 5ug antibody (histones) or 7.5ug antibody (transcription factors/other binding proteins). Protein G Dynabeads were used to select for antibody-bound chromatin in a 4-6 hour incubation. After washing the beads, the crosslinks were reversed and the DNA was purified by phenol-chloroform extraction. 10-30ng of DNA were used for library preparation with end repair, A-tailing, and adaptor ligation (New England Biosciences). The libraries were then size selected for a size of 220-500 bps. The libraries were then indexed using NEBNext Multiplex Oligos for Illumina kit (Cat# E7335S) and 12-15 amplification cycles for ChIP-seq. Adaptors were depleted via size selection on a gel. Library quality and quantity was assessed by Bioanalyzer and multiplexed ten to twelve samples per lane for single-end, 75bp reads on next-gen sequencing on a NEXTseq platform. ChIP antibodies used include: V5 antibody (rabbit polyclonal, Abcam 15828). Total RNA was extracted from at least 1X106 CNCCs at passage 5 on fibronectin from two independent differentiations using Trizol reagent (Invitrogen). 10µg of total RNA underwent two rounds of polyA tail purification with Dynal oligo(dT) beads (Invitrogen) to purify the mRNA. The mRNA was then fragmented with 10X Fragmentation Buffer (Ambion) for exactly 5 minutes and the fragmented mRNA was purified. Then first strand cDNA synthesis was performed with Random Hexamer Primers (Invitrogen) and SuperScript II enzyme (Invitrogen). Next, second strand cDNA synthesis was performed with RNase H (Invitrogen) and DNA Pol I (Invitrogen), and cDNA was purified with a QIAquick column (Qiagen). 30ng of cDNA was used for library preparation, with end repair, A-tailing, and adaptor ligation (New England Biosciences). The double stranded cDNA was then prepared for sequencing using NEBNext Multiplex Oligos for Illumina kit (Cat# E7335S) and only 10-12 amplification cycles for RNA-seq. Adaptors were depleted via a size selection with magnetic beads (Agencourt XP). Library quality and quantity was assessed by Bioanalyzer and multiplexed four to six samples per lane for single-end, 75bp reads on next-gen sequencing on Illumina HiSeq 2500 or NEXTseq platform.
GEO
GSM3955233
GEO Accession
GSM3955233