GSM1978841: PM193mat; Homo sapiens; Bisulfite-Seq
submitter states "custom" (no  meaningful entry) DNeasy Blood and Tissue Kit DNA extraction Parallel PCR reactions to assay 151 imprinted differentially methylated domains (Supplementary material in manuscript) with a final concentration of 1xHotStar PCR buffer, 0.2mM dNTPs, 1U HotStar taq and 0.4uM each primer pair and 2ng DNA were performed in a 15uL reaction volume in 384-well plates.  The plates were prepared on the Agilent Bravo Workstation.  The cycling conditions included a 15 minute 95°C denaturation, followed by 30 cycles of 95°C for 30 seconds, 50°C for 30 seconds, 72°C for 90 seconds, with a 5 minute final extension at 72°C and were performed on the C1000 Touch Thermal Cycler.  PCR products were pooled for each sample and SPRI-purified using Sera-Mag carboxylate-modified Magnetic SpeedBeads as per manufacturer’s recommendations.  A-overhang addition was done using 1x NEB buffer 2, 0.2mM dATP, 12.5U Klenow fragment (3’->5’ exo-) in a 25uL reaction incubated at 37°C for 30 minutes.  Following SPRI purification, Illumina adapters were ligated using the Quick ligation kit in a 40uL reaction.  Following another SPRI purification, libraries were amplified (11 cycles) and barcoded using the Sanger 8-base index in a 50uL reaction containing: 1x High Fidelity buffer, 1U Phusion High Fidelity taq, 0.2mM dNTPs, 0.1uM each primer .  Final libraries were SPRI purified and concentration and quality were assessed by the 2100 Bioanalyzer system and Kapa library quantification.  The 48 barcoded libraries were pooled and multiplexed.
GEO Accession
GSM1978841