GSM4257738: mutant_hESC_library_CTRL_for_Definitive_Endoderm_undifferentiated_rep_1; Homo sapiens; OTHER
At the end of differentiation assay, cells were harvested and genomic DNA was extracted with a Blood & Cell Culture DNA Midi Kit (QIAGEN). genomic DNA was extracted with Blood & Cell Culture DNA Midi Kit (QIAGEN) according to manufacturer's instructions.  The region containing the sgRNA integration was amplified with the following primers that also contain overhang sequences compatible for Nextera DNA library preparations (Illumina): 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNGGCTTTATATATCTTGTGGAAAGGACG – 3' (forward) and 5' - GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACGGACTAGCCTTATTTTAACTTGC - 3' (reverse). Total genomic DNA for each sample was divided into 50 µl PCR reactions with 4 µg DNA input. PCR settings were described previously. After the purification of the 170 base pair product, a second PCR reaction was performed using Nextera adapter primers in order to generate Nextera DNA library according to manufacturer's instructions (Illumina). DNA libraries were sequenced using NextSeq 500 (Illumina) with 75 bp single-end reads.
GEO
GSM4257738