GSM1383851: PrEC NOMe-Seq; Homo sapiens; Bisulfite-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Bisulfite-Seq
Antigen
Bisulfite-Seq
Cell type
Cell type Class
Prostate
Cell type
PrEC
Tissue
prostate
Lineage
epithelial
Description
prostate epithelial cell line
Attributes by original data submitter
Sample
source_name
PrEC
cell line
PrEC
chip antibody
N/A
Sequenced DNA Library
library_strategy
Bisulfite-Seq
library_source
GENOMIC
library_selection
RANDOM
library_construction_protocol
Cells were trypsinized and centrifuged for 3 mins at 500g, then washed in ice-cold PBS and resuspended in 1mL ice-cold Nuclei Buffer (10mM Tris,pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1mM EDTA and 0.5% NP-40,plus protease inhibitors) per 5x106 cells and incubatedon ice for 5 min. Nuclei were recovered by centrifugation at900g for 3 min and washed in Nuclei Wash Buffer (10mM Tris,pH 7.4, 10mM NaCl, 3mM MgCl2 and 0.1mM EDTA containing protease inhibitors). Freshly prepared nuclei (2x105 cells) were resuspended in 1X M.CviPI reaction buffer (NEB), then treated with 150U of M.CviPI (NEB; 50,000U/mL) in 15µL 10X reaction buffer, 45µL 1M sucrose and 0.75µL SAM in a volume of 150uL. Reactions were quenched by the addition of an equal volume of Stop Solution (20nM Tris-HCl [pH 7.9], 600mM NaCl, 1% SDS, 10mM EDTA, 400µg/ml Proteinase K) and incubated at 55°C overnight. DNA was purified by phenol/chloroform extraction and ethanol precipitation. Genomic DNA (2 μg) was sonicated using a Covaris instrument to an average molecular weight of 150 bp. Achievement of the desired size range was verified by Bioanalyzer analysis (Agilent Technologies). Fragmented DNA was repaired to generate blunt ends using the END-It kit (Epicentre Biotechnologies) according to manufacturer’s instructions. Following incubation, the treated DNA was purified using AmpureX beads from Agencourt. Magnetic beads were employed for all nucleic acid purifications in subsequent steps. Following end repair, A-tailing was performed using the dA-tailing module according to manufacturer’s instructions (New England Biolabs). Adapters with a 3’‘T’ overhangs were then ligated to the end-modified DNA. Modified Illumina paired-end (PE) adapters were used. Ligation was carried out using ultrapure, rapid T4 ligase (Enzymatics) according to manufacturer’s instructions. The final product was then purified with magnetic beads to yield an adapter-ligation mix. Prior to bisulfite conversion, bacteriophage lambda DNA that had been through the same library preparation protocol described above to generate adapter-ligation mixes was combined with the genomic sample adapter ligation mix at 0.5% w/w. Adapter-ligation mixes were then bisulfite converted using the Zymo DNA Methylation Gold kit (Zymo Research) according to the manufacturer’s recommendations. Final modified product was purified by magnetic beads and eluted in a final volume of 20μL. Amplification of one-half the adapter-ligated library was performed using Kapa HiFi-U Ready Mix under the following conditions: 98°C for 2 min, followed by 4 cycles of 98°C for 30 s then 65°C for 15 s and 72°C for 1 min with a final extension for 10 min in 50μL total reaction volume. The final library product was examined on the Agilent Bioanalyzer then quantified using the Kapa Biosystems Library Quantification kit according to manufacturer’s instructions. Optimal concentrations to get the right cluster density were determined empirically.