title: GSM5393387: MDA-MB-231 SMARCA4-KO rep1; Homo sapiens; RNA-Seq
library_construction_protocol: The short guide RNA (sgRNA) targeting exon 4 of the human SMARCA4 gene (NM_001128849) was designed by CRISPR design website (https://design.synthego.com). In brief, the 25bp DNA oligos containing the 20 bp target sequence and BsmbI sticky end were annealed and inserted into the lentiCRISPR-v2 plasmid digested with BsmbI (NEB Cat# 0580S). The sgRNA sequence for generating is as follows: Exon 4 SMARCA4 forward: 5'- ATGGTCCCTCTCGCAGCCCA -3' and reverse: 5'- TGGGCTGCGAGAGGGACCAT -3'. To prepare lentivirus particles, cloned lentiCRISPR-v2 plasmid were co-transfected with lentiviral packaging vector pCMV-VSV-G and pCMV-dR8.2 into HEK293FT cells. The viral supernatants were harvested 48h after transfection and filtered through a 0.45-μm pore size filter. Cells were infected for 48h and selected for at least 2 days with puromycin (2ug/ml). For KO validation, genomic DNA from SMARCA4 sgRNA transduced cells was extracted using the DNA Mini Kit (Qiagen) according to manufacturer's instructions and amplified using primers the sgRNA target site. The PCR products were analyzed using TIDE (Tracking of Indels by Sequence Decomposition). TruSeq Stranded mRNA sample Preparation Guide, Part #15031047 Rev. E