title: GSM4368946: Cap_Meth 2 rep3; Homo sapiens; Bisulfite-Seq
library_construction_protocol: For cap meth seq DNA was extracted with Dneasy kit for qiagen (Cat 67506), For RNA seq RNA was isolated with RNeasy kit (Cat 74134). ATAC seq was performed as described previously (Corces, R., Trevino, A.E., Hamilton, E.G., Greenside, P.G., Sinnott-Armstrong, N.A., Vesuna, S., Satpathy, A.T., Rubin, A.J., Montine, K.S., Wu, B., et al. (2017). An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods 14, 959–962.) For RNA seq: Prior to analysis, read adapters and low quality ends were removed using Trim Galore v. 0.4.0. Reads were aligned against hg19 using Tophat v. 2.0.11. Read counts per gene were obtained through htseq-count v.0.6.1p2 in the mode “intersection_nonempty”.    Gene read counts were normalized using the TMM method available from the edgeR_3.24.3 package. For ATAC seq: ATAC samples were preprocessed according to the ENCODE ATAC-seq pipeline. Single-end reads were aligned to the hg19 genome using Bowtie2 with the --local parameter, reads with MAPQ scores < 30 were filtered out with Samtools, duplicates were removed using Sambamba, and TN5 tagAlign shifted files were created. MACS2 was used to call peaks with the following parameters: -p 0.01 --shift -75 --extsize 150 --nomodel -B --SPMR –keep-dup all --call-summits. Peaks were later filtered at a q-value threshold of 0.0001 for further analyses.  Peak counts and sizes for each replicate were calculated using a custom Python script, and Jaccard indices for similarities between called peaks was calculated using BEDTools. For cap meth seq: Following company's protocol (TruSeq-Methyl Capture EPIC, cat # FC-151-1002, Illumina Inc., San Diego, CA) libraries were prepared using 500 ng of genomic DNA. DNA was sonicated using a Covaris LE220 sonicator (Covaris, Woburn, MA) to obtain products of 180–220 bp. DNA was end-repaired, A-tailed and ligated with methylated indexed-adapters to create pre-capture DNA libraries. All pre-capture libraries were hybridized to the EPIC oligos. Hybridized products were subjected to bisulfite conversion. The bisulfite-treated libraries were PCR-amplified. Libraries were sequenced 100 bp paired-end on Illumina NovaSeq-6000
attributes.tag: GEO Accession
attributes.value: GSM4368946