title: GSM8289121: Pancreas, not treated, 7w of age, mouse 1; Mus musculus; RNA-Seq
library_construction_protocol: Based on the commercial protocol, the pancreatic tissue's RNA was extracted using RNeasy Mini Kit (Qiagen #74106). Pancreatic tissue is added to 2 mL of RLT buffer (with 2-Mercaptoethanol, 1:100) and homogenised using a homogeniser (IKA, T10 basic). Homogenizer was washed between the samples with MilliQ water and 70% ethanol to avoid cross-contamination. After tissues were homogenised, 1 volume (2 mL) of 70% ethanol was added. Next, RNeasy Spin columns were loaded with 700μL of the sample and centrifuged for 30 seconds at maximum speed (>13.000rpm). All collected flow-through was discarded. This step was repeated several times. From here, the instructors´ protocol was followed.  The extracted RNA's concentration and sample quality were measured using a spectrophotometer (DeNovix, DS-11). For RNA library preparation the mRNA is enriched by poly(A) capture. RNA is then fragmented by enzyme digestion (or Covaris). cDNA reverse transcription is followed by RNA library preparation. After a library quality control, sequencing is performed using the Illumina NovaSeq 6000 platform, pair-end 150 bp sequencing strategy (short-reads), with a depth of the recommended 20 million read pair per sample. Following data quality control, bioinformatic analysis is performed. Gene set enrichment analysis was performed using the open source GSEA software46