GSM4629789: Subject 2 - medium DNA input (300-1000ng); Homo sapiens; OTHER
DNA was extracted from de-identified PBMC collected from four individuals. Genomic DNA quality was determined by estimating the A260/A280 and A260/A230 ratios by spectrophotometry and concentration by fluorometry. DNA integrity and fragment size were confirmed using a microfluidic chip run on an Agilent Bioanalyzer. To assess the reproducibility of MC-seq by DNA quantity, DNA samples from each participant were profiled in triplicate times with high (>1000ng), medium (300-1000ng) and low (150-300ng) DNA input. In total, 12 DNA samples were measured by MC-seq. Indexed paired-end whole genome sequencing libraries were prepared using the SureSelect XT Methyl-Seq kit (Agilent, part#G9651B). Genomic DNA was sheared to a fragment length of 150-200 bp using focused acoustic energy delivered by the Covaris E220 system (Covaris, part#500003). Fragmented sample size distribution was determined using the Caliper LabChip GX system (PerkinElmer, Part#122000). Fragmented DNA ends were repaired with T4 DNA Polymerase and Polynucleotide Kinase and “A” base was added using Klenow fragment in a single reaction followed by AMPure XP bead-based purification (Beckman Coulter, part#A63882). The methylated adapters were ligated using T4 DNA ligase followed by AMPure XP bead purification. Quality and quantity of adapter-ligated DNA were assessed using the Caliper LabChip GX system. Samples yielding >350 ng were enriched for targeted methylation sites by using the custom SureSelect Methyl-Seq Capture Library. Hybridization was performed at 65°C for 16 hours using a C1000 Thermal Cycler (BIO-RAD, part# 1851197). Once the enrichment was completed the samples were mixed with streptavidin-coated beads (Thermo Fisher Scientific, part#65602) and washed with a series of buffers to remove non-specific bound DNA fragments. DNA fragments were eluted from beads with 0.1 M NaOH. Unmethylated C residues of enriched DNA were modified by bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research, part#D5005). The SureSelect enriched, bisulfite-converted libraries were PCR amplified using custom-made indexed primers (IDT, Coralville, Iowa). Dual-indexed libraries were quantified by quantitative polymerase chain reaction (qPCR) using the Library Quantification Kit (KAPA Biosystems, Part#KK4854) and inserts size distribution was assessed using the Caliper LabChip GX system. Samples with a yield of ≥2 ng/ul were proceeded to sequencing.
GEO Accession
GSM4629789