GSM5669738: Control 1706; Homo sapiens; RNA-Seq
For bulk RNA-seq, total RNA from individual organoids was isolated using the RNeasy Mini Kit (Qiagen, 74104). Genomic DNA was removed using the Invitrogen TURBO DNA-free kit (Invitrogen, AM1907). After isolation, total RNA from individual organoids of the same treatment was pooled for each library preparation. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, E7645) with starting input of 500ng of total RNA per sample. Ribosomal RNA was removed and mRNA isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, E7490). During library preparation, AMPure XP Beads (Beckman Coulter, A63881) were used for reaction clean ups. NEBNext Multiplex Oligos (dual index primers) (New England BioLabs, E7600) were used in the PCR enrichment step, with 11 cycles. Library quality was assessed by Bioanalyzer and the concentration was determined using the NEBNext Library Quant Kit for Illumina (New England BioLabs, E7630). Libraries were pooled and sequenced on the HiSeq4000 machine (Illumina) at 50 base pair length, single ended, to a minimum of 30 million reads per library. For single cell RNA-seq, samples were collected and incubated in 1ml of Accumax (Sigma, A7089) with 400 μg DNase I and 15 μM actinomycin D at 37°C for 20 min with gentle agitation. At 5 min intervals, the sample tubes were flicked, and after 15 min pipetted 10 times, followed by a final pipetting 10 times at 20 min. Clumps were allowed to settle and supernatant collected, to which 100 μl media was added to inactivate Accumax. Samples were then spun at 300xg for 5min. Dead Cell Removal kit and MACS column (Miltenyi, 130-090-101) were used to remove dead cells before another spin at 300g for 5min. Cells were resuspended in an appropriate volume of 0.04% BSA in PBS and loaded on the 10X Chromium system (10X Genomics). Library preparation was performed using the 10X chromium and single cell RNA-seq libraries were prepared according to manufacturer's instructions using the 10X Genomics Chromium Single Cell 3' Library & Gel Bead Kit v3.1 (10X Genomics) workflow. The Chromium Controller was run according to the protocol producing the single cell gel beads in emulsion mixture. Reverse transcriptase reaction and subsequent amplification were carried out on a C1000 Touch Thermal Cycler (Biorad) and libraries were quality tested using a 2100 Bioanalyzer Instrument (Agilent). Samples were pooled together and sequenced on one lane of SP flowcell of the Novaseq sequencer (Illumina).