title: NextSeq 500 sequencing; RNA-seq of mouse intestinal organoids treated with interferon-gamma
design: RNA-seq of mouse intestinal organoids treated with interferon-gamma
library_construction_protocol: Organoids were collected by removing culture media from the tissue culture well. Matrigel constructs holding the organoids were theen dissolved in ice-cold PBS, and the organoids were centrifuged at 1200 rpm for 5 minutes at 4 ºC to form a cell pellet which was used for downstream RNA extraction. The organoids were maintained in Advanced Dulbecco's Modified Eagle Medium/F12 (Thermo Fisher) supplemented with penicillin/streptomycin, 10 mmol/L HEPES, Glutamax, 1x N2 supplement (Thermo Fisher), 1× B27 supplement (Thermo Fisher), 1.25 mmol/L N-acetyl-l-cysteine (Sigma-Aldrich). Wild-type organoids were further supplemented with 50 ng/mL recombinant murine epidermal growth factor (Thermo Fisher), 500 ng/mL RSPO1 (R&D systems), and 100 ng/mL noggin (PeproTech). AKPT and KPN organoids were further supplemented with 50 ng/mL recombinant murine epidermal growth factor (Thermo Fisher) and 100 ng/mL noggin (PeproTech). Organoids were plated on DAY 0 and the media was supplemented with 1 ng/mL of recombinant mouse interferon-gamma protein (PeproTech) on DAY 3. Samples were collected 24 h later. RNA was extracted using the RNeasy Micro Kit from Qiagen (74004) and DNase treatment was performed using the DNA-free kit from Life Technologies (AM1906). 3'RNAseq libraries were prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD (Lexogen, Austria). RNA input varying from 100-1000ng and 14-20 PCR cycles were used. Library quality and quantity were assessed using the HS 1000 DNA TapeStation (Agilent) and DNA High Sensitivity Qubit Assay (ThermoFisher) respectively.