GSM9238266: SCLC13; Homo sapiens; Bisulfite-Seq
FFPE sections were deparaffinized in mineral oil. Briefly, 800µL mineral oil (Fisher Scientific, #AC415080010) and 180µL Buffer PKD were mixed with the sections, Proteinase K was added for tissue digestion, and the sample was incubated at 56 °C for 15 min. Phase separation was encouraged with centrifugation, and the aqueous phase was chilled 3 min to precipitate RNA. After centrifugation for 15 min at 20,000 g, RNA-containing supernatant was removed for extraction, while DNA remained in the pellet. Nucleic acids were subsequently extracted using the AllPrep DNA/RNA Mini Kit (QIAGEN, #80204) according to the manufacturer's instructions. RNA was eluted in nuclease-free water and DNA in 0.5X Buffer ATE. After PicoGreen quantification (ThermoFisher, #P11496) and quality control by Agilent BioAnalyzer, 170–750 ng of genomic DNA was sheared using a LE220-plus Focused-ultrasonicator (Covaris, #500569). Samples were cleaned using Sample Purification Beads from the TruSeq Methyl Capture EPIC LT Library Prep Kit (Illumina, #FC-151-1002) according to the manufacturer's instructions with modifications. Briefly, samples were incubated for 5 min after addition of SPB, 50 µL RSB was added for resuspension, and resuspended samples were incubated for 2 min. Sequencing libraries were prepared using the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) without PCR amplification. Post-ligation cleanup proceeded according to Illumina's instructions with 110 µL Sample Purification Mix. After purification, 3–4 samples were pooled equimolar and methylome regions were captured using EPIC oligos. Capture pools were bisulfite converted and amplified with 11–12 cycles of PCR. Pools were sequenced on a NovaSeq 6000 or HiSeq 4000 in a 150/150 bp or 100 bp/100 bp paired end run, using the NovaSeq 6000 S4 Reagent Kit (300 Cycles) or HiSeq 3000/4000 SBS Kit (Illumina). The average number of read pairs per sample was 51 million.