GSM9510043: Custom Panel2 HCMS, HAdCC Donor3, EE7000 mRNAOnly, 2; Homo sapiens; OTHER
From each PHH donor, 4 x 105 µL aliquots of lysate were processed for DNA extraction. From each HAdCC and HSEC donor, 4 x 63 µL aliquots of lysate were mixed with 42 µL of additional LBE buffer per aliquot and processed for DNA extraction. For all samples, lysate aliquots were transferred to separate technical replicate wells of a 0.5 mL deep well plate and mixed with Beckman Coulter patented SPRI paramagnetic beads to bind total DNA from the cell lysate. DNA-bound beads were then pelleted with a plate magnet, and the supernatants were removed and discarded before DNA-bound beads were treated with RNase in Beckman Coulter WBA buffer, pelleted with a plate magnet, and the supernatants removed. The retained DNA-bound beads were then washed twice with 70% ethanol. gDNA was eluted from the paramagnetic beads in 10 mM Tris pH 8.0, all twelve technical replicate wells combined and transferred to a clean storage plate. The first step of the Hybrid Capture Methylation Sequencing (HCMS) workflow was the preparation of EM-seq libraries using Twist NGS Methylation Detection System. Samples were normalized in 10 mM Tris pH 8.0 to generate aliquots containing 200 ng of gDNA in 50 µL. Samples were then mechanically sheared on a Covaris R230 Focused Ultrasonicator to a size range of 350 – 450 bp and at least 5% of the samples were evaluated on a D1000 HS ScreenTape to ensure target size range was reached. The second step of the HCMS workflow was enrichment of DNA fragments for sequencing. Target-enrichment for subsequent methylation sequencing was accomplished through a hybridization reaction in which biotinylated DNA probes were used to capture regions of interest from the whole genome EM-seq library. The biotinylated DNA probe panel and capture reagents were supplied by Twist Biosciences and the workflow was performed following the manufacturer's recommendations. Briefly, the hybridization reaction was carried out by first drying down 8-plex library pools in a mix of the custom biotinylated DNA probe panel, reaction buffer, and universal blocking oligos, which prevent library fragments from annealing to each other at their adapter sequences. The dried reactions were then resuspended in prewarmed hybridization buffer (65˚C) and incubated on a thermocycler for a hybridization time of 16 hours. Following hybridization, biotinylated DNA probes bound to target gDNA sequences were enriched using streptavidin beads. Streptavidin beads bound to the probe-library complex were pelleted on a magnet and unbound (i.e., off panel) fragments removed through a series of heated washes. The final bead slurry was resuspended in water before performing a PCR amplification to enrich the captured library fragments. Following amplification, libraries were purified with Twist Total Purification beads. Final library concentrations were determined by using the Quant-iT High Sensitivity DNA kit on the VICTOR Nivo plate reader. Library fragment size was assessed on a High Sensitivity DNA 1000 ScreenTape on the Agilent 4200 Tapestation instrument. Libraries were pooled in an equimolar manner and, to ensure proper quantitation of sequenceable library molecules, qPCR was also performed using the NEBNext Library Quant Kit for Illumina.  Enriched EM-seq libraries were shipped to SeqCenter for sequencing on a NovaSeq X. Sequencing used a 2 x 151 bp configuration and a targeted sequencing depth of >200 M read-pairs per library.