title: RNA-seq of intestinal organoids treated with the complement C5a receptor 1 (C5aR1) antagonist PMX205 with or without irradiation
design: RNA-seq of intestinal organoids treated with the complement C5a receptor 1 (C5aR1) antagonist PMX205 with or without irradiation
library_construction_protocol: After treatment, organoids were harvested by direct incubation in 350 ul RLT Plus lysis buffer (Qiagen RNeasy Plus Micro kit) in each well for 5 minutes. At least 30 mg of mouse small intestine was collected from a 5 week-old, wild-type, male C57BL/6 mice for optimal cell yield and organoid establishment. The tissue was transferred into a 50 ml conical centrifuge tube with 30 ml of PBS for short-term storage on ice until use. In a cell culture hood, tissues were transferred to a 10 cm Petri dish. Using small scissors, the intestinal section was cut open lengthwise as such that the lumen of the intestine was facing up and was then minced into smaller pieces of ∼2 mm. Pieces were rinsed twice with cold PBS and transferred into a 50 ml conical centrifugee tube filled with 30 mL of 10% FBS/PBS solution. The tube was shaken forcefully serval times and the intestinal pieces left to settle by gravity to the bottom of the tube. The 10% FBS/PBS solution was replaced with 25 ml of HBSS/EDTA solution (1.86 g of dehydrated EDTA (Sigma-Aldrich) and 500 ul of 10M NAOH (Sigma-Aldrich) in a 500 ml HBSS solution without Ca2+ and Mg2+ (Thermo Fisher Scientific)). The tube was incubated in a 37°C water bath for 10 minutes and shaken forcefully several times every 2-3 minutes. The crypts were released into the supernatant by shaking the tube. The supernatants containing the isolated crypts were collected and the procedure was repeated twice. Each fraction was filtered through a 150 µm cell strainer to remove debris and for crypts enrichment and 1ml of each fraction was examined under the microscope to check for culturable crypts (it is common for the first fraction to have minimal crypts isolated). 25 ml of 10% FBS/PBS solution was added to the fraction with higher numbers of isolated crypts and centrifuged at 1200 rpm at 4°C for five minutes. The pellet was washed with 25 ml of 10% FBS/PBS solution and spinned down again. The supernatant was removed carefully and the crypt containing pellets was resuspended in cold Cultrex™ UltiMatrix Reduced Growth Factor (RGF) Basement Membrane Extract (BME) (Bio-Techne). Approximately 250 crypts per 20 ul of BME were seeded in each well of a pre-warmed 48-well plate. The BME was then solidified by a 20-minute incubation in a 37°C and 5% CO2 cell culture incubator and overlaid with 500 µl of complete mouse organoid media (Advanced Dulbecco's Modified Eagle Medium/F12 (Thermo Fisher) supplemented with GlutaMAX, 10mM HEPES, 100ug/mL Primocin, 1X N-2 supplement, 1X B-27 supplement, 0.1% BSA, 25% R-spondin-1 conditioned medium, 100 ng/mL mouse EGF, 100 ng/mL mouse Noggin, and 3 μM CHIR 99021) with 10 μM Y-27632 (Tocris). 10 µM Y-27632 ameliorate organoids establishment and apoptosis survival. Complete media was subsequently refreshed every two days.   Organoids were usually passaged every 5–10 days by a mechanical approach using Gentle Cell Dissociation Reagent (GCDR) (STEMCELL Technologies). The mechanical method is recommended for expansion of organoids and before freezing as it is less harsh. Briefly, medium was aspirated and organoids were incubated with 500 ml GCDR at room temperature for a minimum of 10 minutes. Using a p1000 pipette, the BME domes were scraped off the surface of the plate and transferred with the GCDR into a 15 mL conical centrifuge tube (Multiple wells can be split together into one tube). The gel/GCDR mix were pipetted several times to break up the organoids. For maintenance passage, 10–15 rounds of pipetting are sufficient to dissociate the organoids to fragments that will quickly regenerate complete organoids. The tube was filled with basal medium up to 10 ml, and centrifuged at 1200 rpm for 5 min. The pellets were resuspended in BME and re-seeded at an appropriate ratio. Organoids were biobanked in FBS (Thermo Fisher Scientific) containing 5% DMSO (SigmaAldrich). Organoids were plated on Day 0 and the media was supplemented with 5 μg/ml PMX205 (Tocris #5196) dissolved in 20% ethanol/water on Day 1. Control organoids were provided with media supplemented with an equal amount of vehicle control (20% ethanol/water). Cells were pre-treated for 1 hour before irradiation with either 0 or 9 Gy irradiation. Samples were collected either 24 h or 48 h after irradiation with 9 Gy. RNA was extracted using the Qiagen RNeasy Plus Micro Kit according to the manufacturer protocol. RNA quality and concentration were measured with Agilent Bioanalyzer 2100 (Agilent) and Nanodrop (Thermo Fisher Scientific) respectively. Library construction was performed by Novogene UK. 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. Non-directional libraries were prepared by second strand cDNA synthesis using dTTP and were ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Libraries were checked for quantification with Qubit and real-time PCR and for size distribution detection with an Agilent Bioanalyzer.