GSM4505212: 0 hrs untreated; Homo sapiens; RNA-Seq
Single cells were diluted in PBS, supplemented with 0.04% bovine serum albumin up to a final concentration of 100 cells/µl. Using a microfluidic PDMS device (Nanoshift), single cells were co-encapsulated in droplets with barcoded beads (Chemgenes Corporation, Wilmington, MA) at a final concentration of 120 beads/uL. Droplets were collected for 15 min/sample. After droplet breakage, beads were harvested, washed, and prepared for on bead mRNA reverse transcription (Maxima RT, Thermo Fisher). Following an exonuclease I (New England Biolabs) treatment for the removal of unused primers, beads were counted, aliquoted (2000 beads/reaction, equals ~100 cells/reaction), and pre-amplified by 13 PCR cycles (primers, chemistry, and cycle conditions identical to those previously described in Macosko et al., 2015). PCR products were pooled and purified twice using 0.6x clean-up beads (CleanNA). Prior to tagmentation, cDNA samples were loaded on a DNA High Sensitivity Chip on the 2100 Bioanalyzer (Agilent) to ensure transcript integrity, purity, and amount. For each sample, 1 ng of pre-amplified cDNA from an estimated 1000 cells was tagmented by Nextera XT (Illumina) with a custom P5 primer (Integrated DNA Technologies). Single cell libraries were sequenced in a 100 bp paired-end run on the Illumina HiSeq4000 using 0.2 nM denatured sample and 5% PhiX spike-in. For priming of read 1, 0.5 µM Read1CustSeqB was used (primer sequence: GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC).
GEO Accession
GSM4505212