title: GSM8491283: TasteOrganoids_CQVsLPM_LPM_Sample1; Sus scrofa; RNA-Seq
library_construction_protocol: Organoid harvesting from 6-well/12-well plates was done using the Cultrex™ Organoid Harvesting Solution (Cat 3700-100-01). Briefly; working on ice the cell culture media was gently aspirated and each well was washed with PBS (2ml/1ml). 1ml of the organoid harvesting solution was added to each well, the domes gently dislodged using a cell scraper/lifter and incubated for 1hr at 4C0 on a rocking shaker (Reliable Scientific Inc.) at a speed of 50, for BME digestion. Post incubation, the organoids were spun down and washed three times with 1ml PBS. Each spin down was done at 400g for 4mins at 4C0. The palleted organoids from each well were lysed in 600µl of RLT Plus lysis buffer (Qiagen) with 6µl of ß-mercaptoethanol (Sigma Life Science Cat# 63689-100ML-F) and homogenized using ~200µl of 1.0mm Zirconia (BioSpec Cat# 11079110ZX) beads with a single 30 second, 6,000 rpm cycle on a Precellys24 homogenizer (Bertin Corp). Homogenized solution was spun down for 20mins at 8000rpm prior to RNA isolation using RNEasy Plus Mini Kit (Qiagen Cat# 74134/74136) according to manufacturer's protocol. FP were homogenized in the Extraction Buffer provided in the Arcturus™ PicoPure™ RNA Isolation Kit (Applied Biosystems Cat# 12204-01) using the same bead based method described above. RNA extraction followed the recommended Arcturus™ PicoPure™ RNA Isolation Kit protocol without performing the DNAse step since TaqMan probes are at the junctions of gene exons Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification.