GSM2652194: shCont_RbSox2_2; Mus musculus; OTHER
First, plasmids harboring shRNA-GFP were transfected into mouse NeuPCs, and GFP-positive cells were sorted into 10% FBS/PBS. Subsequently, cells were cross-linked with 1% formaldehyde at room temperature for 10 min. After sonicating cells by a M220 Focused-ultrasonicator (Covaris), lysate containing about 350,000 cells was aliquoted.　Chromatin was incubated withα-Sox2 (Cell Signaling) antibody at 4℃ overnight. Subsequently, protein A Dynabeads were transferred to tubes and incubated at 4℃ for 2 hours, and beads were washed. Beads were resuspended in tagmentation reaction solution, incubated for 10 min at 37℃, then cooled on ice. Beads were washed, chromatin was decross-linked, and DNA was extracted and purified by Ampure XP beads (Beckman). A DNA library was generated using 2xKapa HiFi HotStart Ready Mix and Nextera custom primers. qPCR was used to determine the number of PCR cycles, and 11-12 cycles in total were used. The library was analyzed on Tape station analyzer (Agilent Technologies) and followed by deep sequencing with the Illumina’s Hiseq2500 at SE50.
GEO Accession
GSM2652194