title: GSM3670709: LE_B_7; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Blood from mothers were taken from a fasting blood sample collected in T3. Blood from infants were sampled with a neonatal heel prick 48h after birth. Blood from both mother and infant was collected in EDTA tubes and stored at -80°C.   Genomic DNA was extracted from whole blood using DNeasy Blood and Tissue Kit (QIAGEN, Germany) according to the manufacturer's protocol. Yield and quality was assessed by Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Denmark ) and NanoDrop (Thermo Fisher Scientific). Multiplexed reduced representation bisulfite sequencing (RRBS) was performed as previously described. Briefly, genomic DNA was incubated with Msp1 (NEB, Germany) restriction enzyme overnight for fragmentation. Adenylation was performed using dNTP (NEB) and Klenow fragment (NEB) followed by an AMpure Bead clean-up (Beckman Coulter, Denmark) and ligation to TruSeq adapters (Illumina). Twelve ligated samples were pooled and subjected to bisulfite conversion using EZ DNA Methylation Kit (Zymo Research, Germany). The library pool was amplified by PCR (2 min 95 °C (30 s 95 °C, 30 s 65 °C; 45 s 72 °C) × 20 cycles; 7 min 72 °C) with PfuTurbo Hotstart DNA polymerase (Agilent, Denmark). PCR products were purified using the AMPure Bead clean-up. Libraries were sequenced on a HiSeq 2500 (Illumina) at the Danish National High-Throughput DNA Sequencing Centre, on a 50 bp single-end sequencing mode.
attributes.tag: GEO Accession
attributes.value: GSM3670709