GSM2074774: 6-C11+D1 CRISPR/Cas9 and a homologous donor template Experiment_3; Homo sapiens; OTHER
Genomic DNA was extracted using the Invitrogen PureLink Genomic DNA Mini Kit (CA#K182002 ) according to the manufacturer's instructions Genomic DNA samples were initially amplified outside the donor template with primers OuterFwd 5'-ATGCTTAGAACCGAGGTAGAGTTT-3' and OuterRev 5'-CCTGAGACTTCCACACTGATG-3'. 1ul of the PCR was run on a 1% agarose gel to confirm band size. 1ul of PCR product was then used for the "inner" PCR with primers HBB Fwd 5’ – ACACGACGCTCTTCCGATCTNNNNGGCAGAGCCATCTATTGCTT – 3’ and HBB Rev 5’ – GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTCTCCTTAAACCTGTCTTG – 3’ to amplify the specific 198 bp region of HBB surrounding the sickle mutation along with primers to add Illumina adapters and indexes. 1ul of the second PCR product was run on a 2% agarose gel to confirm the band size was accurate. Quantification of individual samples was completed using densitometry and samples were pooled at equimolar ratios before being PCR purified using Ampure Beads. The final library was quantified using droplet digital PCR for the Illumina adapters and diluted to 10nM before being run on an Illumina HiSeq machine
GEO Accession
GSM2074774