GSM8463339: Neocortex Chd8+/- 1; Mus musculus; Bisulfite-Seq
DNA was extracted using DNeasy DNA extraction (Qiagen, 69504) kit. DNA concentrations were checked using Qubit Fluorometry and 100ng of DNA was used for RRBS library preparation using the Premium RRBS kit (Diagenode, C02030032). DNA was digested using MspI overnight at 37˚C after which adapters were ligated. Prior to quantification by qPCR, fragments were selected based on fragment size using AMPure XP beads (Beckman Coulter, A63880). For qPCR quantification, 1µl of size-selected library, LUNA Universal qPCR Master Mix (New England Biolabs, M3003), 3.5µl of nuclease free H2O, and 0.5µl primer mix (from the RRBS kit) transferred to 96-well PCR plate. Libraries were pooled based on the formula (17 x 2-ΔCt , where ΔCt = Ctmax –Ctsample). Pooled libraries were topped up with nuclease free H2O before clean up with AMPure XP beads. Bisulfite conversion was performed using BS Conversion reagent (from the Premium RRBS kit), before enrichment PCR, quality control and clean-up.