GSM8147021: Evf1 GAPDH TSTS CHIRPseq; Mus musculus; OTHER
The method described in (Chu et al., 2011) was followed, and modified for embryonic day 13.5 ganglionic eminences (13.5GEs). For each sample ~16 E13.5GEs were dissected for each genotype (Evf1/2+/+, Evf1/2TS/TS) in ice cold L15, pipetted to produce a single cell suspension, pelleted for 4min, X 800 RCF, resuspended in PBS, pelleted for 4min, X 800 RCF, and resuspended in 5mL of 1% glutaraldehyde. Cells were cross-linked for 10 minutes, quenched with 500 µL of 1.25 M glycine for 5 minutes, pelleted for 5 min X 2000 RCF, 4°C, washed with PBS, pelleted for 5 min X 2000 RCF for 5min, 4°C, resuspended 1mL 4°C PBS per 8 embryos, pelleted X 2000 RCF for 3 minutes at 4°C, supernatant aspirated, and cell pellets flash frozen in liquid nitrogen and stored at -80°C. Libraries were created with NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1), and 10M reads from PE100 sequenced on NOVAseq. Raw sequencing reads for ChIRPdatasets were aligned using bowtie2 (Langmead and Salzberg, 2012) mapper with the following settings '--local --very-sensitive-local --no-mixed --no-discordant --phred33 -I 10 -X 700'.The datasets weremappedagainst the mm9 version of the mouse genome.