title: GSM2111395: Cell__number_36; Mus musculus; RNA-Seq
library_construction_protocol: Mice were perfused with 20ml HBSS (Gibco), the pancreas was extracted and adipose tissue removed. Four solutions were prepared, including D solution: 1mg/ml Collagenase Type CLS IV supplemented with 0.25% BSA (Sigma-Aldrich), R solution: 1% BSA dissolved in PBS, C solution: 4% BSA in PBS and I solution: 0.1% BSA in PBS. The tissue was chopped into small pieces and incubated in 10ml of D solution at 37°C for 30 minutes. The digestion product was filtered through a 70μm cell strainer (Islets of Langerhans were thereby removed). 10ml of R solution was pipetted on the cell strainer. A quarter of the filtered cell suspension was gently transferred on top of 6ml C solution to achieve layer separation of the liquids. Acini were spun down at 50g for 2 minutes and washed with C solution and I solution successively. Purified acini were treated with 2ml Accutase (Sigma-Aldrich) for 5min to acquire acinar cell suspension containing single cells and clusters of acinar cells Single cell RNA seq library preparation protocol was based on the SMART seq2 protocol by Picelli et al., 2014 with following modifications: Acinar cells were isolated as described in the section “Acinar Cell Isolation and Culture” and resuspended in DPBS without Ca2+ and Mg2+ (Beckman Coulter). Cells were collected in a volume of 0.5µl and transferred to a reaction tube containing 4µl of 6M Guanidine-HCl (Sigma-Aldrich) and 0.1% (vol/vol) Triton X-100 (Sigma-Aldrich). The tube was immediately transferred into liquid nitrogen and kept there for the duration of cell collection. Next, 2.2x RNA SPRI beads (Beckman Coulter) was added directly to the lysis buffer and incubated for 5 min at room temperature. The beads were washed twice with 70% Ethanol. Air-dried beads were resuspended in a solution containing 2µl H20, 1 µl oligo-dT primer and 1µl dNTP mix (primer and nucleotides used as in 4). 24 cells contained ERCC Spike-In RNAs (1:10 000 – Mix2, Ambion) Mix in addition to primer and nucleotides. Beads were incubated for 3 min at 72°C and reverse transcription and PCR (19 cycles) were performed as described in Picelli et al., 2014. PCR product was cleaned up using 0.8x DNA SPRI beads (Beckman Coulter) and air dried beads were resuspended in 15µl H2O. Quality of cDNA library was assessed for each cell on a high sensitivity DNA Bioanalyzer chip. Subsequent steps (tagmentation, amplification, multiplexing) were done as previously described in Llorens-Bobadilla et al., 2015
attributes.tag: GEO Accession
attributes.value: GSM2111395