title: GSM4503623: 293T prostate cancer cells, DNA, replicate3; Homo sapiens; OTHER
library_construction_protocol: 1st round PCR primers contain target-specific sequences flanking the 30-bp randomer barcode and Illumina adaptor sequences, producing a product of 215 bp (see Supplementary Table 7f).  The 1st round PCR reaction was performed using 2x Phusion Flash Mastermix (ThermoFisher) in a 50 µL reaction. The PCR reaction consisted of 5 µL of DNA or cDNA template, 2 µL of forward primer (10 µM), 2 µL of reverse primer (10 µM) and 25 µL of Phusion Flash Mastermix. Thermal cycling conditions were at 95°C for 3 min, 20 cycles of (98°C for 10 sec, 60°C for 30 sec, 72°C for 30 sec), followed by 72°C at 5 min. A small portion (3 µL) of the PCR products and negative controls were run on a 1.5% agarose gel for visual inspection. The 1st round PCR products were purified using a 0.8x AMPure XP (Beckman Coulter) cleanup following the manufacturer's protocol with 80% ethanol Following cleanup, 4 µL of the purified 1st round PCR product was used as a template in the 2nd round PCR reaction.  The forward primer contains the Illumina adaptor sequence, as well as the flow cell attachment sequence, the reverse primer contains an 8-bp index between the adaptor sequence and flow cell attachment sequence. The 2nd round PCR reaction was carried out in a 50 µL reaction similarly, using Phusion Flash Mastermix (ThermoFisher), with 5 µL of each forward and reverse primer (0.5 µM).  Thermal cycling conditions were at 95°C for 3 min, 8 cycles of (95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec), followed by 72°C at 5 min. PCR products were purified using a 0.8x AMPure XP (Beckman Coulter) cleanup following the manufacturer's protocol with 80% ethanol. A sample (3 µL) of the purified PCR products were run on a 1.5% agarose gel for visual inspection. Each sample was quantified by qPCR using the KAPA Library Universal Quantification kit (KAPA Biosystems) according to the manufacturer's instructions and pooled in equimolar amounts for multiplex sequencing. The final pool was denatured and diluted to a loading concentration of 7.5pM as per Illumina protocol. The PhiX control library (Illumina) was spiked in at 20% to add diversity for improved cluster imaging.
attributes.tag: GEO Accession
attributes.value: GSM4503623