GSM3732854: Dropseq_4; Homo sapiens; RNA-Seq
For each sample, 1-1.5 g of tissue was homogenized by mincing with scissors into smaller pieces (~0.5 mm2/piece). Prior to tissue digestion, lung homogenates were cleared from excessive blood by addition of 35 ml of ice-cold PBS, followed by gentle shaking and tissue collection using a 40μm strainer. The bloody filtrate was discarded. The tissue was transferred into 8 ml of enzyme mix consisting of dispase (50 caseinolytic U/ml), collagenase (2 mg/ml), elastase (1 mg/ml), and DNase (30 µg/ml) for mild enzymatic digestion for 1 hour at 37°C while shaking. Enzyme activity was inhibited by adding 5 ml of PBS supplemented with 10% FCS. Dissociated cells in suspension were passed through a 70μm strainer and centrifuged at 300g for 5 minutes at 4°C. The cell pellet was resuspended in 3 ml of red blood cell lysis buffer and incubated at room temperature for 2 minutes to lyse remaining red blood cells. After incubation, 10 ml of PBS supplemented with 10% FCS was added to the suspension and the mix was centrifuged at 300g for 5 minutes at 4°C. The cells were taken up in 1 ml of PBS supplemented with 10% FCS, counted using a Neubauer chamber and critically assessed for single cell separation. Dead cells were counted to calculate the overall cell viability, which needed to be above 85% to continue with Drop-Seq. 250,000 cells were aliquoted in 2.5 ml of PBS supplemented with 0.04% of bovine serum albumin and loaded for Drop-Seq at a final concentration of 100 cells/μl. Drop-seq experiments were performed largely as described previously (Pubmed ID: 26000488) with few adaptations during the single cell library preparation. Briefly, using a microfluidic polydimethylsiloxane (PDMS) device (Nanoshift), single cells (100/µl) from the lung cell suspension were co-encapsulated in droplets with barcoded beads (120/µl, purchased from ChemGenes Corporation, Wilmington, MA) at rates of 4000 µl/hr. Droplet emulsions were collected for 15 min/each prior to droplet breakage by perfluorooctanol (Sigma-Aldrich). After breakage, beads were harvested and the hybridized mRNA transcripts reverse transcribed (Maxima RT, Thermo Fisher). Unused primers were removed by the addition of exonuclease I (New England Biolabs), following which beads were washed, counted, and aliquoted for pre-amplification (2000 beads/reaction, equals ~100 cells/reaction) with 12 PCR cycles (primers, chemistry, and cycle conditions identical to those previously described. PCR products were pooled and purified twice by 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 (primer sequence: GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC) was used.
GEO Accession
GSM3732854