GSM3231485: mHDR_control_pool2_sort1_noGFP; Homo sapiens; OTHER
DNeasy Blood & Tissue Kit, eluted in 200 ul H2O. To amplify the barcode from gDNA from the GFP-positive and negative populations were spread over 8-16 reactions containing 250 ug of gDNA each (220K–440K genome equivalents by weight given HeLa triploid genome ~9 pg). Reactions also contained Kapa2G Robust Polymerase (Kapa Biosystems), a primer that annealed to the SV40 promoter 5' of the integrated plasmid and a primer adjacent to the barcode (SV40_F and newpc5bc_nexteraR). PCR was performed using the following conditions [95˚, 5 min; {95˚, 40s, 65˚, 30s, 72˚, 3 min} x ~28 cycles; 72˚, 10 min]. The reactions produce a ~3,700 base amplicon specifically from integrated plasmids. The reactions for each sample were combined and the amplicons were purified by 0.5X Ampure and eluted with 10 mM Tris at 10% of the original reaction volume. 10% of the eluted PCR volume was re-amplified with primers containing sample indexes and Illumina cluster generating sequences (pc5bc_p5_F, nextIndex). Reactions also contained Kapa2G Robust Polymerase and 0.5X SYBR green II (ThermoFisher). PCR reactions are monitored on a Mini-opticon qPCR machine (Bio-Rad) and removed during exponential amplification using the following conditions [95˚, 3 min; {95˚, 20s, 65˚, 30s, 72˚, 20s} x 5-10 cycles]. The PCR reaction produced a 350-base amplicon that was purified using a double-Ampure protocol. First the large DNA fragments were removed by precipitation by addition of 0.6X volume Ampure beads to the reactions. Then the 350-base amplicons were purified from the supernatant using 0.9X volume Ampure beads. The samples were multiplexed and the barcodes and sample indexes were sequenced (single read, pcDNA5_barcodeSeq_F) on a Nextseq 500 High Output 75 base kit.
GEO Accession
GSM3231485