GSM3374608: pcr_wafergen_expt_standard; Homo sapiens; RNA-Seq
Wafergen Illumina libraries at 2 nM concentration were diluted 20 fold and PCR amplified with 1 unit of Phusion and the cell specific primer and Nextera read 2 sequencing primer to specifically amplify the sequences from the selected cells for 14 cycles.   PCR results were confirmed by gel electrophoresis and the remaining sample was purified with Ampure XP beads at a ratio of 1.8x bead/pcr volume. Samples were eluted and added in equal volume to prepared Dynabead M270 streptavidin beads. Beads were incubated for 15 minutes at room temperature with gentle rotation.  Beads were washed 5x in bind and wash buffer and eluted in 20µl of water. 10µl of streptavidin purified input DNA was added to PCR using PEPCRPrimer 1.0 (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT)  and Nextera N703 (CAAGCAGAAGACGGCATACGAGATttctgcctGTCTCGTGGGCTCGG) for 9 cycles. PCR products were ampure purified, confirmed on the Agilent Tapestation D1000, quantified by the Qubit and submitted for sequencing. Read 1 positions 1-11 contain the 11bp cell barcode, and positions 12-22 contain a 10bp UMI. Read 2 contains the cDNA.
GEO Accession
GSM3374608