title: GSM2719769: HCT116 RNA-seq-WT-Rep2; Homo sapiens; RNA-Seq
library_construction_protocol: ATAC-Seq: Cells were washed with cold PBS, collected by centrifugation then resuspended in resuspension buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2). After collection, cells were lysed in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40) and collected before incubating in transposition mix containing Tn5 transposase. ChIP-seq: Cells were harvested and crosslinked in 1% formaldehyde for 10 min before quenching with glycine for 5 min on ice. Cells were pelleted by centrifugation and snap frozen in dry ice before storage at -80˚C. Pellets were thawed on ice and resuspended in rinse buffer 1 (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X100), collected by centrifugation then resuspended in rinse buffer 2 (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Cells were washed and resuspended in shearing buffer before sonication using Covaris E220 (0.1% SDS, 1mM EDTA, pH 8, 10mM Tris HCl, pH 8). For ChIPs using antibodies for histone modifications, 106 cells in 1ml shearing buffer were added to 1ml Covaris tubes and sheared for 12 mins at 140W with 10% duty factor. For FRA1 ChIPs, cells were sheared for 8 mins at 140W with 5% duty factor. DNA was then made up to 1x IP buffer (50 mM HEPES/KOH pH 7.5, 150 mM NaCl , 1 mM EDTA, 1 % Triton X100, 0.1 % DOC, 0.1% SDS, supplemented with protease inhibitors) and 3ug antibody added for overnight incubation with rolling at 4˚C. Antibody bound DNA was recovered using a 1:1 mixture of Protein A and Protein G beads, washed and treated with Proteinase K and RNAse A. Purified ChIP DNA was then used for library generation for ChIP-seq. RNA-Seq: RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research). ATAC-seq: Purified DNA was ligated with adapters (NuGEN), amplified and size selected for sequencing. Library DNA was sequenced with paired end 42 bp reads. RNA-seq: mRNA was isolated using NEBnext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs) with 5 μg input RNA according to the manufacturer’s guidelines. Libraries were prepared for RNA-seq using NEBnext® UltraTM RNA Library Prep Kit for Illumina® following the manufacturer’s instructions. mRNA was fragmented and purified before first strand and second strand synthesis. Double-stranded cDNA was then purified and ends repaired before dA tailing and adapter ligation. After cleanup and size selection, cDNA libraries were amplified and purified before sequencing with single end 50 bp reads. ChIP-seq: 5 ng ChIP DNA was used to prepare libraries using the NuGen Ovation® Ultralow Library System V2 following the manufacturer’s instructions. Briefly, DNA ends were repaired before ligation of adapters for sequencing. Following bead purification, libraries were amplified by PCR then purified and size-selected for sequencing with single end 50 bp reads.