title: GSM4151128: Blastocyst Day 5 Library 7; Homo sapiens; OTHER
library_construction_protocol: When required, embryos were briefly treated with Acidic Tyrode's Solution (Sigma-Aldrich, Belgium) for removal of the zona pellucida. All embryos were washed and subsequently dissociated by gentle trituration in TrypLE Express Enzyme (Life Technologies, Belgium) using glass capillaries. Single blastomeres were washed and manually deposited into 384-well plates containing lysis buffer and Vapor-lock. Plates were subsequently centrifuged at 1,000 rpm for 1 minute and stored at -80 °C until further processing. Single cells were incubated at 50C for 15 hours, 75C for 20 minutes and 80C for 5 minutes. Glucosylation of 5hmC sites followed by dispensing 0.1 L of T4-BGT (NEB), 0.1 L of UDP-Glucose (NEB), 0.05 L of 10X NEB Buffer 4 and 0.25 L of nuclease-free water (final vol 0.5mL). After incubation at 37C for 16 hours, 0.5 L of the following reaction mixture is added: 0.1L of 25g/L Qiagen Protease, 0.05L of 10X NEB Buffer 4 and 0.35 L of nuclease-free water. The plate is then incubated at 50 o C for 5 hours, 75 o C for 20 minutes and 80 o C for 5 minutes. Thereafter, gDNA is digested by the restriction enzyme MspJI by the addition of 0.5 L of the following reaction mixture: 0.02 L of MspJI (NEB), 0.12 L of 30X enzyme activator solution (NEB), 0.05 L of 10X NEB Buffer 4 and 0.31 L of nuclease-free water. The digestion is performed at 37 o C for 5 hours followed by heat inactivation of MspJI at 65 o C for 20 minutes. Next, 0.2 L of cell-specific double-stranded adaptors are added to individual wells and these adapters are ligated to the fragmented gDNA molecules by adding 0.8 L of the following reaction mixture: 0.07 L of T4 DNA ligase (NEB), 0.1 L of T4 DNA ligase buffer (NEB), 0.3 L of 10 mM ATP (NEB) and 0.33 L of nuclease-free water. The ligation is performed at 16 o C for 16 hours. Next, wells containing unique cell-specific adapters are pooled using a multichannel pipette and incubated with 0.8X Agencourt Ampure (Beckman Coulter) beads for 30 minutes, washed twice with 80% ethanol and resuspended in 6.4 L of nuclease-free water. In vitro transcription and Illumina library preparation is performed as described previously in the scAba-seq protocol.
attributes.tag: GEO Accession
attributes.value: GSM4151128