title: GSM4042935: Transitional GFP- Mid Repli-seq rep1; Mus musculus; OTHER
library_construction_protocol: Genomic DNA was isolated from each fraction using SDS-proteinase K buffer and purified by phenol-chloroform extraction. Genomic DNA was fragmented using the Covaris sonicator to get 700 bp average fragment size. The sheared BrdU-labeled DNA from each fraction was immunoprecipitated using 0.5 μg of mouse anti-BrdU antibody followed by 50 μL of precleared-Dynabeads sheep anti-mouse IgG (Invitrogen, 11201D). The immunoprecipitated pellet was digested overnight with Proteinase K and purified by phenol-chloroform extraction. The replication timing libraries were prepared based on Accel-NGS methyl seq library kit (Swift Biosciences, MI, USA) according to the manufacturer's protocol. The BrDU immuno-precipitated DNA was denatured and subjected to Adaptase reaction. This step was followed by an extension reaction with two cleanup steps utilizing Agencourt Ampure XP beads (Beckman Coulter, IN, USA). The elute was subjected to ligation step with a follow-up of Ampure bead mediated purification. Indexing PCR was done with appropriate and unique adaptor primer for an individual sample preparation. PCR program consisted of 98ºC for 30 seconds, then 9 cycles of 98ºC for 10 seconds, 60ºC for 30 seconds and 60ºC for 60 seconds followed by a 4ºC hold cycle. The PCR product was further purified by Ampure beads and eluted out in a 20μl volume using TE buffer provided by the manufacturer. The libraries were checked for size, using Agilent 2200 Tape Station (Agilent, CA, USA) utilizing DNA high sensitivity tape (Agilent, CA, USA). Up to 12 libraries were pooled together after Qubit quantification with Qubit DNA HS assay kit (Thermofisher Scientific, NY, USA) and loaded into Nextseq 500/550 high output cartridge (Illumina, CA, USA) for 75 cycle single end sequencing.
attributes.tag: GEO Accession
attributes.value: GSM4042935