title: GSM5889049: MAC050721_013; Mus musculus; RNA-Seq
library_construction_protocol: Whole ileum was flushed with PBS, the Peyer's patches removed, opened longitudinally, and cut into 1 cm pieces. Three consecutive washes were performed with HBSS supplemented with 10% bovine calf serum, 15 mM HEPES, 5mM EDTA and 1.25 mM DTT at room temperature for 20 min under continuous rotation followed by 20 seconds of vortexing in PBS, pH 7.4, to dissociate intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs). The remaining lamina propria (LP) pieces were transferred to 15 ml RPMI 1640 supplemented with 10% FBS, 50 U/ml penicillin, 50 ug/ml streptomycin and 2 mM L-glutamine with 0.5 mg/ml Collagenase VIII (Sigma Aldrich) warmed to 37oC. Samples were shaken vigorously by hand and placed in a shaking incubator at 220 rpm and 37oC for 15 min with additional manual shaking at 10 min. After 15 min, 35 ml ice-cold complete media was added to stop enzymatic digestion. Samples were washed twice with FACS buffer (PBS pH 7.4 plus 1% FBS and 2 mM EDTA) and filtered with 100 um and 70 um strainers. 50% of the IEC fraction was combined with the LP fraction. Cells were stained with Zombie NIR viability dye and AF488 anti-CD45 antibody. Live, CD45+ and CD45- singlets were sorted on a MoFlo Astrios Cell Sorter (Beckman Coulter) into RLT buffer containing 1% beta-mercaptoethanol, flash frozen on dry ice and stored at -80oC. RNA extraction was performed using an RNeasy Plus Micro Kit (Qiagen) according to the manufacturer's instructions. RNA samples were checked for purity using NanoDrop™ One Spectrophotometer (Thermo Scientific) and analyzed for integrity using 4200 TapeStation (Agilent). Relative levels of remaining DNA were checked by dual RNA/DNA measurements using Qubit fluorometer (Invitrogen). DNA amounts did not exceed 10% of the total amount of nucleic acid. Libraries for Illumina sequencing were prepared using 10-20 ng of total RNA per sample. Library preparation was performed with the Universal Plus mRNA-Seq library preparation kit (Tecan/NuGen, 0520-A01), as written in the product manual (NuGen, M01485 v5). In brief, RNA underwent poly-A selection, enzymatic fragmentation, and generation of double-stranded cDNA using a mixture of oligo(dT) and random priming. The cDNA underwent end repair, ligation of dual-index adaptors, strand selection, and 16 cycles of PCR amplification. The number of cycles was determined by qPCR on a small aliquot of the unamplified libraries. All intermediate purification steps, and final library purification was carried out using Agencourt AMPure XP Beads (Beckman Coulter A63881). Final amplified libraries were measured with the Qubit 1X dsDNA HS Assay Kit (Invitrogen, Q33231), and fragment size distribution was confirmed to be approximately 360 bp using the D5000 ScreenTape assay (Agilent, 5067-5588, 5067-5589). Concentration of the final library pool was confirmed by qPCR and subjected to test sequencing in order to check sequencing efficiencies and adjust accordingly proportions of individual libraries. The pool was purified with the Agencourt AMPure XP Beads (Beckman Coulter A63881), quantified by qPCR using KAPA Library Quantification Kit and sequenced on a NovaSeq 6000 S4 flow cell, 2x150 bp, approximately 30 M clusters per sample, at the University of Illinois Roy J. Carver Biotechnology Center High-Throughput Sequencing and Genotyping Unit.