title: GSM4151089: E14 B6 Library 1; Mus musculus; OTHER
library_construction_protocol: Cells were isolated using hyaluronic acid (Sigma) and trypsin (Life Technologies) and sorted into 384-well plates containing lysis buffer and Vapor-lock. 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: GSM4151089