GSM5068664: Arid1a_knockout_rep3 [RNA-seq]; Mus musculus; RNA-Seq
The pancreas of 8-week-old p48-Cre and Arid1afl/fl;p48-Cre mice were harvested in ice-cold HBSS, dissociated by surgical scissors in a 10 cm cell culture dish filled with 5 ml cold HBSS and centrifuged at low speed (300 rpm, 5 min) to remove blood vessels and adipose tissue. The remaining explant was digested in 5 ml solution 2 at 37 ºC for 20 min, pipetted every 2 min using a 10 ml pipette. The tissue-containing tube was washed with 10 ml solution 1 and centrifuged (300 rpm, 5 min) before adding a second time of solution 2 and cultivated at 37 ºC for 10 min. Next, tissue suspension was transferred into a new 50 ml tube by filtering through a 100 µm cell strainer and the tissue fragments were ground using a syringe plunger. The remaining cells were collected by washing the mesh with two times of 10 ml solution 1 and pelleted. Recovering was achieved by easing the acinar cells in 5 ml solution 3 containing 10% FCS for 1 hour at 37 ºC and total RNA was extracted by TRIzol method. Libraries were prepared from 500 ng RNA utilizing Truseq RNA Library Prep Kit v2 following manufacturer's instructions. mRNA was captured by RNA purification beads and eluted. The first strand and second strand cDNA were synthesized and the overhangs of the fragments were repaired into blunt ends. The 3' end was added with a single adenine to prevent mutual ligation during the following adapter conjugation. Finally, the DNA fragments with both flow-cell-hybridization adapters were amplified and the library was quantified by Agilent Bioanalyzer 2100. Size selection was optional if the main population of the library exceeds the appropriate size for sequencing or small fragments such as primer-dimers exist.