GSM1824700
GSM1824700: hs_17y_Cortex_WhiteMatter; Homo sapiens; Bisulfite-Seq
To extract genomic DNA from paraffin block for human cortex gray and white matter, xylene was first added to these blocks, vortexed for 1 minutes and gentle shaked at RT for 5-15min. The supernatant was discarded after centrifugation, the pellets were washed with absolute ethanol twice to remove xylene, dried them on bench. 1ml lysis solution was added  (50 mM Tris, 25 mM EDTA, 100 mM NaCl, 0.5% Tween-20/SDS, pH 8), inverted every 30 minutes until the pellets were completely lysed,  40 μl 20mg/ml proteinase K was added, Incubated overnight at 55 ºC.  After 1-hour 90 ºC incubation, 5ul RNase cocktail was added and incubated at 37 ºC for 30minutes. Phenol-cholorophorm method was used to extract the DNA from samples Hairpin bisulfite-seq library construction:  10ug genomic DNA of each sample was spiked with 0.02% unmethylated Lambda DNA (Promega) and sonicated to 200bp fragments with Covaris. After purification with PureLink® PCR Purification Kit (Invitrogen), DNA fragments were subjected to MseI and MluCI digestion (NEB) for 1h at 37°C, after purification, the elute DNA was subjected to end repair with the end repair enzyme mix (T4 DNA polymerase, Klenow enzyme, T4 PNK; NEB), dA tailing using Klenow 3’-5’ exo- (NEB) with purification at each step. Ligation to Biotin-modified hairpin adapter (5’P - GGCCAGCTGCA AG/iBiodT/GAAGCAGCTGGCCT-3', IDT) were performed using T4 DNA ligase (NEB) at 16°C for 2h with molar ratio 1:1. Then hairpin adapter and Illumina TruSeq adapter (Illumina) was added and continued ligation overnight with the final ratio of DNA: hairpin adapter: Illumina TruSeq adapter =1:10:10.  After purification by Ampure XP beads (Agencourt), DNA fragments were pulled down by Dynabeads® MyOne™ Streptavidin C1 beads (Invitrogen). Dynabeads® MyOne™ Streptavidin C1 beads were boiled at 95°C for 15min, and then subjected to Bisulfite conversion using the EpiTect Bisulphite Kit (Qiagen). After desulfonation and purification, the single-stranded uracil-containing DNA was subjected to 16 cycles of PCR reaction with Illumina TruSeq PCR primers (IDT) and 2.5 U Pfu TurboCx Hotstart DNA polymerase (Agilent). After purification by Ampure XP beads, size selection of 400-700 bp fragments was conducted with 2% agarose Pippin Prep cassettes (sage science). Libraries were sequenced using Illumina HiSeq 2000 or MiSeq.
GEO
GSM1824700
GEO Accession
GSM1824700