GSM4705551
GSM4705551: MCF7 sgNTR3; Homo sapiens; OTHER
Total genomic DNA was extracted from MCF7 and MDA-MB-231 cells using QIAamp DNA Mini Kit.Total RNA was extracted from MCF7 cells using the RNeasy Mini kit. Two rounds of PCR were employed to prepare the next-generation-sequencing (NGS)-ready sgRNA libraries with the KAPA HiFi HotStart ReadyMix (Roche # KK2602). The first-round PCR was conduced for 16 cycles, using 40 g of input genomic DNA from each replicate at D0 or D21 as a template. The PCR product of different samples was pooled and 20 l of the mixed product was used as a template for the second-round PCR, which was conducted for 12 cycles to incorporate Illumina barcode sequences. The final PCR product was purified from 2% agarose gel with QIAquick Gel Extraction Kit. Concentration of different libraries was measured using the Qubit dsDNA HS (High Sensitivity) Assay Kit (Thermo # Q32851) on a Qubit Fluorometer (Thermo fisher). Two ug of RNA was used for RNA-seq library construction with TruSeq Stranded mRNA Library Prep kit (Illumina # 20020594).
GEO
GSM4705551
GEO Accession
GSM4705551