GSM8827098: LuCaP_105; Homo sapiens; MeDIP-Seq
DNA from the LuCaP PDXs was extracted using the DNeasy Blood and Tissue Kit (Qiagen). Genomic DNA was sheared using a Covaris Sonicator E220 and AMPure XP beads (Beckman Coulter) were used to size select 150-250 bp DNA fragments. Library preparation was performed using KAPA HyperPrep Kit (KAPA Biosystems) according to the manufacturer's protocol. We then performed end-repair, A-tailing, and ligation of NEBNext adaptors (NEBNext Multiplex Oligos for Illumina kit, New England BioLabs). Libraries were digested using the USER enzyme (New England BioLabs). λ DNA, consisting of unmethylated and in vitro methylated DNA, was added to prepared libraries to achieve a total amount of 100 ng DNA. Methylated and unmethylated Arabidopsis thaliana DNA (Diagenode) was added for quality control. MeDIP was performed using the MagMeDIP kit (Diagenode) following the manufacturer's protocol. Samples were purified using the iPure Kit v2 (Diagenode). Success of the immunoprecipitation was confirmed using qPCR to detect recovery of the spiked-in Arabidopsis thaliana methylated and unmethylated DNA. KAPA HiFi Hotstart ReadyMix (KAPA Biosystems) and NEBNext Multiplex Oligos for Illumina (New England Biolabs) were added to a final concentration of 0.3 uM and libraries were amplified as follows: activation at 95°C for 3 minutes, amplification cycles of 98°C for 20 seconds, 65°C for 15 seconds, 72°C for 30 seconds, and a final extension of 72°C for 1 minute. Samples were pooled and sequenced (Novogene Corporation, CA) on Illumina HiSeq 4000 to generate 150 bp paired-end reads.