GSM1465033: DKO1 RNA polymerase II ChIP-seq rep2; Homo sapiens; ChIP-Seq
Whole Genome Bisulfite Sequencing (WGBS): Genomic DNA was collected from HCT116 and DKO1 cells using a Qiagen QIAeasy DNA mini kit. ChIP-seq: Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. RNA-seq: RNA was collected from cells using Trizol (Life Technologies Catalog#15596018). Whole Genome Bisulfite Sequencing (WGBS): Genomic DNA (2 μg) was sonicated using a Covaris to an average molecular weight of 150 bp.  Achievement of the desired size range was verified by Bioanalyzer (Agilent) analysis. Fragmented DNA was repaired to generate blunt ends using the END-It kit (Epicentre Biotechnologies, Madison, WI) according to manufacturer’s instructions.  Following incubation, the treated DNA was purified using AmpureX beads from Agencourt.  In general, magnetic beads were employed for all nucleic acid purifications in the following protocol.  Following end repair, A-tailing was performed using the NEB dA-tailing module according to manufacturer’s instructions (New England Biolabs, Ipswich, MA). Adapters with a 3’ ‘T’ overhang were then ligated to the end-modified DNA.  For whole genome bisulfite sequencing, modified Illumina paired-end (PE) adapters were used in which cytosine bases in the adapter are replaced with 5-methylcytosine bases.   Depending on the specific application, we utilized either Early Access Methylation Adapter Oligos that do not contain barcodes, or the adapters present in later versions of the Illumina DNA Sample Preparation kits, which contain both indices and methylated cytosines.  Ligation was carried out using ultrapure, rapid T4 ligase (Enzymatics, Beverly, MA) according to manufacturer’s instructions.  The final product was then purified with magnetic beads to yield an adapter-ligation mix. Prior to bisulfite conversion, bacteriophage lambda DNA that had been through the same library preparation protocol described above to generate adapter-ligation mixes was combined with the genomic sample adapter ligation mix at 0.5% w/w.  Adapter-ligation mixes were then bisulfite converted using the Zymo DNA Methylation Gold kit (Zymo Research, Orange, CA) according to the manufacturer’s recommendations. Final modified product was purified by magnetic beads and eluted in a final volume of 20 ul. Amplification of one-half the adapter-ligated library was performed using Kapa HiFi-U Ready Mix for the following protocol: 98° 2’; then six cycles of  98° 30”, 65° 15” , 72° 60”; with a final 72° 10’ extension, in a 50 ul total volume reaction. The final library product was examined on the Agilent Bioanalyzer, then quantified using the Kapa Biosystems Library Quantification kit according to manufacturer’s instructions.   Optimal concentrations to get the right cluster density were determined empirically but tended to be higher than for non-bisulfite libraries.  Libraries were plated using the Illumina cBot and run on the Hi-Seq 2000 according to manufacturer’s instructions using HSCS v 1.5.15.1.  Image analysis and basecalling were carried out using RTA 1.13.48.0, deconvolution and fastq file generation were carried out using CASAVA_v1.7.1a5. RNA-seq was performed for HCT116 and DKO1 in replicate. RNA was collected from cells using Trizol (Life Technologies Catalog#15596018) and  paired-end libraries were prepared using the Illumina TruSeqV2 Sample Prep Kit (Catalog #15596-026), starting with 1 ug total RNA. Libraries were sequenced on an Illumina Hi-Seq 2000. ChIP-seq: protocol was described in Comparison of sample preparation methods for ChIP-chip assays. O'geen H et.al Biotech 2006, 41:577–580.