title: GSM2027535: hESC MBD-seq R1_MBD; Homo sapiens; MBD-Seq
library_construction_protocol: genomic DNA was isolated using qiagen blood and tissue kit. Methyl binding domain (MBD) reactions. MBD and control reactions were performed according to the manufacturers’ protocol (MethylMiner Methylated DNA Enrichment Kit; ME10025, Invitrogen). Isolated genomic DNA was sonicated and gel purified at a range of 100-300 bp. 3.5 ug was used for methyl enrichment using the single fraction elution protocol. Five to ten nanogram of MBD purified DNA was end-repaired, a single adenosine was added and ligated to Illumina adaptor oligonucleotides (TruSeq DNA sample prep Kit, Illumina). Sonicated  14  genomic DNA at 1% of MBD reaction input was used for input library preparation. 13 cycles of PCR was performed with universal primer P1.0 (5’AATGATACGGCGACCACCGAGATCTACACTCTTTCC CTACAC GACGCTCTTCCGATCT) and a barcoded index primer (5’CAAGCAGAAGACGGCATACG AGAT NNNNNNGTGACTGGAGTTC).Both RNA-seq and MBD-seq library templates were prepared for sequencing using cBot cluster generation system (Illumina) with TruSeq PE Cluster V3 Kit. The sequencing run was performed with paired end mode of 100 cycles of read 1, 7 cycles of index read, and 100 cycles of read 2 using Illumina HiSeq 2500 platform with TruSeq SBS V3 Kits. Sequencing library preparation and reactions were conducted at City of Hope’s integrative genomics core