GSM7103819: RH4, sgNFYC, JUN, rep1; Homo sapiens; OTHER
Cells were trypsinized and counted on Invitrogen Countess Automated Cell Counter. 1x107 cells were used per viewpoint and processed in batches. Cells were fixed in 2% formaldehyde fixation buffer at RT for 10 minutes and quenched with 0.125 M glycine on ice. Cells were then washed in PBS and lysed in 1 ml lysis buffer (50mM TRIS, 150mM NaCl, 5mM EDTA, 0.5% NP-40, 1% Triton X-100 + protease inhibitors) for 10 min on ice. Batch-processed cells were then split into individual conditions for downstream processing. First digestion was performed with DpnII and the second digestion with Csp6I. Four separate PCR reactions for each viewpoint were carried out using 150-200 ng 4C template input material.The following primers were used (R = reading primer, NR = non-reading primer): ERRFI1_R TACACGACGCTCTTCCGATCTGCCTGATAGTTAGACACTGATC; ERRFI1_NR ACTGGAGTTCAGACGTGTGCTCTTCCGATCAGGACCTTTCCTCCATATTC; JUN_R TACACGACGCTCTTCCGATCTCAAGCGTGTAGGCGATC; JUN_NR ACTGGAGTTCAGACGTGTGCTCTTCCGATCAAACTTAAGTCCCCTTAGGC; PEG3_R TACACGACGCTCTTCCGATCTGGTAGCATTAGATGTGACGATC; PEG3_NR ACTGGAGTTCAGACGTGTGCTCTTCCGATCGGCTGTCTGCCCTAATGC; SIX1_R TACACGACGCTCTTCCGATCTGGGTTGTTGAGACCAGATC; SIX1_NR ACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTTCCAATAAGAAGTGACCA. Reactions were pooled and purified using Ampure XP beads, eluting in 35 µl milliQ water. Ten µl purified PCR1 product was used as input for a second PCR to obtain full Illumina adapter sequences using the universal forward primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and a barcoded reverse primer (CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGCT) with a 6 nucleotide barcode (XXXXXX). PCR reactions were then purified using a Roche high pure PCR purification kit. Pooled 4C reactions were purified once more with Ampure XP beads and sequenced on the Illumina NextSeq 2000 platform using the single end SE75 bp mode of sequencing.