title: GSM8705242: RNA - Pancreas - 28 days post-cerulein injection #3; Mus musculus; RNA-Seq
library_construction_protocol: Pancreata from mice were collected upon sacrifice; after collection, first, a small piece of each clamp frozen pancreas piece was pulverized by using a liquid nitrogen cooled Cellcrusher. Nuclei were isolated from tissue powder employing a protocol for isolating nuclei from RNAse-rich tissue that uses citric acid, previously described (Tosti et al., Gastroenterology, 2019). In short, one small spoon of tissue powder (5-10 mg approximately) was suspended into 1 mL of pre-cooled S25 buffer, composed by 1.5M sucrose (Sigma-Aldrich – S7903), 250mM citric acid (Sigma-Aldrich – C0759), 0.1% Hoechst 33258 (Life Technologies – H3569), inside a glass douncer tissue grinder (DWK Life Sciences - 357358). With 5 strokes of a tight pestle every 3 minutes for 10 minutes, nuclei were released into the buffer. Subsequently, the lysate was transferred through a 35 μm strainer into a 1.5 mL Eppendorf tube and centrifuged at 4°C for 5 min at 500 g. Afterwards, the supernatant was removed and the pellet was resuspended in 500 μL S25 buffer. To remove clearly visible cell debris, a density centrifugation was performed, by carefully adding 500 μL S88 buffer, composed by 1.5M sucrose and 250mM citric acid, to the bottom of the Eppendorf tube followed by centrifugation at 4°C for 10 min at 1000 g. After removal of the supernatant, the nuclei were resuspended in 250 μL S25 buffer, their integrity was checked and they were counted with a CountessTM II FL Automated Cell Counter (Thermo Fisher Scientific). The samples were spun down again at 4°C for 5 min at 500 g, filtered through a 10 μm strainer and resuspended in 100μL resuspension buffer. The nuclei suspension was diluted to load 10000 nuclei per sample onto the 10x Genomics Chromium controller. Libraries were generated with the Multiome Kit (10x Genomics - PN231000283) following the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression user guide and the finished libraries were sequenced on an Illumina NovaSeq6000 (S4, paired-end). The provided fastq files are provided for simplicity as concatenation of 4/6 sequencing runs, as indicated, as named according to 10X cellranger conventions.