GSM3878565: GeCKO-Trim28_SORT_T2; Mus musculus; OTHER
After 3 days after the fourth sorting, 2 x 10^5 cells were collected and genomic DNA was extracted using Wizard Genomic DNA Purification kit (Promega). PCR of the virally integrated guides was performed on sgDNA at the equivalent of approximately 2000 cells per guide in 48 parallel reactions using KOD-FX neo (TOYOBO, Japan) in a single-step reaction of 22 cycles. Primers are listed here: forward primer, AATGATACGGCGACCACCGAGATCTACACTCTTTC CCTACACGACGCTCTTCCGATCTNNNNNNNN(1–8-bp stagger) GTGGAAAGGACGAAACACCG; reverse primer, CAAGCAGAAGACGGCATACGAGATNNNNNNNN GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTGGGCGATGTGCGCTCTG, 8-bp index read barcode indicated in italic. PCR products from all 48 reactions were pooled, purified using PCR purification kit (Qiagen, Hilden, Germany) and gel extracted using the Gel extraction kit (Qiagen, Hilden, Germany). Resulting libraries were deep-sequenced on Illumina HiSeq platform with a total coverage of >8 million reads passing filter per library.
GEO Accession
GSM3878565