ST blastocysts were freed from their zonae pellucidae and plated on mEF feeder layers on 4-well culture dishes for 5-7 days at 37°C, 3% CO2, 5% O2, and 92% N2 in medium (DMEM/F12 with 10% FBS, 10% KSR, 0.1 mM nonessential amino acids, 1 mM l-glutamine, 0.1 mM β-mercaptoethanol, 5 ng/ml basic fibroblast growth factor, 10μM ROCK inhibitor) as previously described8. Attached blastocyst outgrowths were manually minced into small pieces, re-plated onto fresh plates and maintained in knockout DMEM medium (Invitrogen) supplemented with 20% KSR, 0.1 mM nonessential amino acids, 1 mM l-glutamine, 0.1 mM β-mercaptoethanol, penicillin-streptomycin and 4 ng/ml bFGF for further propagation and analysis.
Extracted molecule
genomic DNA
Extraction protocol
Extraction of genomic DNA from ESC lines was performed using Qiagen Puregene DNA isolation kit.
Label
C-Bio and A-DNP
Label protocol
200ng of genomic DNA was whole-genome amplified in an overnight reaction at 37¡C using Multi-Sample amplification 1 mix (MA1), Multi-Sample amplification master mix (MA2) and Random Primer Mix (MSM). After incubation the amplified DNA was fragmented with fragmentation solution (FMS), precipitated with Precipitation solution (PM1) and resuspended in Resuspension, hybridization, and wash solution (RA1).
Hybridization protocol
RA1 resuspended DNA was loaded onto BeadChips arrays. After overnight incubation at 48¡C, single-base extension and allele-specific staining was performed on the Illumina Hyb chamber Rack system.
Scan protocol
After allele-specific staining BeadChip arrays were coated with XC4/ethanol , dried for 1 hour and scanned on a HiScan system (Illumina).
Description
3243ST-ES2
Data processing
Genomic DNA extracted from ESC line was genotyped using the Infinium Assay with the Illumina CytoSNP-850Kv1.1 BeadChip platform.
