title: GSM5343616: Calu3 Gattinara screen 1/2 initial population; Homo sapiens; OTHER
library_construction_protocol: NucleoSpin Blood XL kit as per the manufacturer's instructions For PCR amplification, gDNA was divided into 100 μL reactions such that each well had at most 10 μg of gDNA. Plasmid DNA (pDNA) was also included at a maximum of 100 pg per well. Per 96 well plate, a master mix consisted of 150 μL DNA Polymerase (Titanium Taq; Takara), 1 mL of 10x buffer, 800 μL of dNTPs (Takara), 50 μL of P5 stagger primer mix (stock at 100 μM concentration), 500 μL of DMSO, and water to bring the final volume to 4 mL. Each well consisted of 50 μL gDNA plus water, 40 μL PCR master mix, and 10 μL of a uniquely barcoded P7 primer (stock at 5 μM concentration). PCR cycling conditions were as follows: an initial 1 min at 95 °C; followed by 30 s at 94 °C, 30 s at 52.5 °C, 30 s at 72 °C, for 28 cycles; and a final 10 min extension at 72 °C. PCR primers were synthesized at Integrated DNA Technologies (IDT). PCR products were purified with Agencourt AMPure XP SPRI beads according to manufacturer's instructions (Beckman Coulter, A63880). Prior to sequencing the sample was quantitated by qPCR and diluted to 2nM. 5 µL of the  sample was then further diluted and denatured with 5 µL 0.1N NaOH and 490 µL HT1 buffer (Illumina). Samples were sequenced on a HiSeq2500 HighOutput (Illumina) with a 5% spike-in of PhiX.
attributes.tag: GEO Accession
attributes.value: GSM5343616