GSM1592464: AT2 WGBS Donor 2 -Run1; Homo sapiens; Bisulfite-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Bisulfite-Seq
Antigen
Bisulfite-Seq
Cell type
Cell type Class
Lung
Cell type
AT2
NA
NA
Attributes by original data submitter
Sample
source_name
Whole genome bisulfite sequencing
cell type
purified AT2 cells
individual
Donor 2
Sequenced DNA Library
library_strategy
Bisulfite-Seq
library_source
GENOMIC
library_selection
RANDOM
library_construction_protocol
DNA was isolated from freshly isolated primary AT2 cells using the Illustra TriplePrep Kit. WGBS libraries were made by the USC Epigenome Center. 4 μg of sample genomic DNA was sonicated using a Covaris S2 to an average molecular weight of 150 bp. Achievement of the desired size range was verified by Bioanalyzer (Agilent) analysis. Fragmented DNA was repaired to generate blunt ends using the END-It kit (Epicentre Biotechnologies, Madison, WI) according to manufacturer’s instructions. Following incubation, the treated DNA was purified using AmpureX beads from Agencourt. In general, magnetic beads were employed for all nucleic acid purifications in the following protocol. Following end repair, A-tailing was performed using the NEB dA-tailing module according to manufacturer’s instructions (New England Biolabs, Ipswich, MA). Adapters with a 3’ ‘T’ overhang were then ligated to the end-modified DNA. For whole genome bisulfite sequencing, modified Illumina paired-end (PE) adapters were used in which cytosine bases in the adapter are replaced with 5-methylcytosine bases. Ligation was carried out using ultrapure, rapid T4 ligase (Enzymatics, Beverly, MA) 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, Orange, CA) according to the manufacturer’s recommendations. Final modified product was purified by magnetic beads and eluted in a final volume of 20 ul. Amplification of one-half the adapter-ligated library was performed using Kapa HiFi-U Ready Mix for the following protocol: 98° 2’, Then six cycles of: 98° 30”, 65° 15”, 72° 60”, with a final 72° 10’ extension, in a 50 ul total volume reaction. 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 but tended to be higher than for non-bisulfite libraries. Libraries were plated using the Illumina cBot and run on the Hi-Seq 2000 according to manufacturer’s instructions using HSCS v 1.5.15.1. Rep 1 underwent Paired End 100 cycling; rep 2 underwent Paired End 75 cycling.