GSM7026355: pancreas, Ad-M3C, day 06, r3; Mus musculus; RNA-Seq
Adenovirus infected pancreas was perfused with 2 to 3 mL collagenase P (0.8 mg/mL) through the common bile duct and digested at 37 oC for 15 to 20 min. Digestion was stopped by adding 15 - 20 mL DMEM medium containing 10% FBS, 1% PenStrep and 1% HEPES (quenching buffer, QB). The suspension was filtered with 70 μm cell strainer. The filtrate was spun for 3 min at 300 g, 4 oC, resuspend the pellet with ice-cold DPBS to clear FBS twice dissociated the pellet into single cells using 1 mL TrypLE Express at 37 oC for 15 - 20 min, and mixed the solution up and down gently by P1000 pipette during the digestion process to improve the proportion of single cells. After digestion, the cell suspension was spun for 3 min at 300 g, 4 oC, resuspended with FACS buffer (DPBS with 0.5% BSA) and filtered with 40 μm strainer to obtain single cell suspension. The high viability single cells were labeled by Calcein Blue AM (1 mg/mL), which was added into cell suspension directly at the ratio of 1:2000. The single cells labeled by mCherry and Calcein Blue AM were sorted into cell lysis buffer in 96-well plates through Moflo Astrios 4 lasers flow cytometry. The collected samples were fast frozen at the mixture of dry ice and absolute ethanol. scRNA-seq libraries were constructed according to the Smart-seq2 protocol except that oligo d(T) primers comprising 16-bp cell barcode sequence and 9-bp molecular barcode sequence were used to allow sample pooling and molecular counting. In addition, part of the TruSeq read2 sequence was used to replace the ISPCR sequence in the oligo d(T) primer thus to be compatible with Illumina sequence platform. Cells in lysis buffer were denatured, reverse transcribed in the presence of template switching oligos, and pre-amplified by adding both ISPCR and ISPCR-read2 primers with 24 PCR cycles. cDNA sequences of cells with different barcodes were then pooled together and purified using 0.8× AMPure XP beads (Catalog No. A63881, Beckman, Brea, CA). Homemade Tn5 enzyme was used to tagment cDNA. Final amplification was processed using P7-index primers (TruSeq) and P5-index primers (Nextera). Sequencing libraries were purified with 0.6× AMPure XP beads twice and with 1× AMPure XP beads once, and then sequenced on Illumina HiSeq X10 platform with default parameters.