GSM5285681: AK9134_het_P3_d5; Mus musculus; RNA-Seq
RNA from organoids was extracted using Trizol (Sigma-Aldrich). mRNA was enriched from 500 ng total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer's instructions. The polyadenylated RNA fraction was eluted in 15ul 2x first strand cDNA synthesis buffer (NEBnext, NEB). After chemical fragmentation by incubating for 15 min at 94°C the samples were directly subjected to the workflow for strand specific RNA-seq library preparation (Ultra II Directional RNA Library Prep, NEB). In short, the first and second strand synthesis followed an end repair and A-tailing of the fragments that were ligated with the universal NEB hairpin loop adapter. Finishing ligation with incubation at 20°C for 15 minutes, cDNA was subjected to size selection by AMPure XP beads (Beckman Coulter) to remove fragments longer than 800 bp and shorter than 200 bp. Remaining adapters were depleted with an additional XP bead purification with equal amounts of sample and XP bead solution. During the PCR enrichment with 12 cycles, dual unique index primers were incorporated carrying the sequence for i7 and i5 tails (Primer 1: AAT GAT ACG GCG ACC ACC GAG ATC TAC AC XXXXXXXX ACA TCT TTC CCT ACA CGA CGC TCT TCC GAT CT, Primer 2: CAA GCA GAA GAC GGC ATA CGA GAT XXXXXXXX GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T; X represents the different barcode sequences). After two more AMPure XP bead purifications (in a ratio of 1:0.9), libraries were quantified using the Fragment Analyzer (Agilent).