GSM8447294: H460,rep1,4Cseq; Homo sapiens; OTHER
Cells were cross-linked with 4% PFA in PBS pH7.4 and lysed in cold lysis buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 5mM EDTA, 0.5% Tergitol NP-40, 1% Triton® X-100 and 1X Complete protease inhibitors  First restriction enzyme digestion was done using NlaIII (NEB, R0125L) followed with ligation by T4 DNA ligase (NEB, M0202L). Reverse crosslinking was using 15 uL Proteinase K (20mg/mL) at 65℃ overnight and genomic DNA purified by ethanol precipitation method. Second restriction enzyme digest was done on the ligated products using DpnII (NEB, R0543S) and finally ligated to form 4C-templates which were purified MercK Millipore AmiconTM ultra-15 centrifugal filter units. 4C library was prepared from the 200ng template using Q5 Polymerase  and HPLC purified P7(read1) and P5(read2) illumina merged long sequencing primers (IDT) which spanned the viewpoint (TBXT promoter). (Viewpoint hg38 TBXT promoter iF 5'-GGCTTTCCCGCGGACATG-3' and iR(5'-TTATTTCCGTCCATTTCCCTC-3'). PCR done on 98℃ for 30 seconds, 30 cycles of 98℃ for 10 seconds, annealing temperature gradient of 55℃ for 30 seconds and 72℃ for 30 seconds. 4C library PCR products were purified using High Pure PCR Product Purification Kit (Roche, 11 732 668 001) and Agencourt, AMPure XP reagent. Libraries were sequenced as on Illumina platform HiseqXten