GSM8862621: Ileal muscularis ILC2s; cKO; replicate 3; H. polygyrus (7 d.p.i.); Mus musculus; RNA-Seq
Mice were euthanized by CO2 narcosis and perfused with PBS through the left ventricle. The distal 12-centimeters of small intestines were dissected on ice, cut longitudinally along the mesenteric border, and laid flat (luminal side down) on C-fold towels dampened with ice-cold holding buffer (HBSS without Ca2+ and Mg2+, 5% FCS, 10mM HEPES) on a metal tray in an ice bucket. Muscularis propria were isolated from lamina propria by stereomicroscope-aided microdissection performed on an ice-cold metal block lined with buffer-dampened C-fold towels, rinsed 2-3x in holding buffer to limit epithelial cell contaminants, and processed separately from lamina propria samples. The lamina propria samples were stripped of epithelial cells by sequential washing and occasional vortexing in ice-cold IEC isolation buffer with DTE (HBSS without Ca2+ and Mg2+, 5% FCS, 10mM HEPES, 5mM EDTA, 1mM DTE) followed by 2-3 additional washes of the same buffer without DTE, and finally rinsed with PBS without EDTA. Both the muscularis and lamina propria samples were separately minced with scissors and digested in digestion buffer (HBSS with Ca2+ and Mg2+, 5% FCS, 10mM HEPES, 100 μg mL-1 DNase I, Liberase TL enzyme) for 15-30 minutes at 37°C in flasks with stir-bars spinning at 550 rpm. Liberase TL concentrations of 100 μg mL-1 and 200 μg mL-1 were used for muscularis and lamina propria samples, respectively. After digestion, samples were gently triturated with 3-5 passes through an 18G needle attached to a syringe and single-cell suspensions were strained through 40μm nylon meshes, washed, and further processed for FACS purification. ILC2s were defined as viability dye-negative, singlet, CD45+, CD90.2+, Lineage(Ter119, NK1.1, B220, CD19, CD3e)-negative, KLRG1+ cells and sorted on a FACSAria II sorter (BD Biosciences). Between 215 to 2,000 FACS-purified ILC2s (depending on the cellular recovery from each tissue) were sorted directly into 5 μL of Buffer TCL (Qiagen) supplemented with 1% β-mercaptoethanol (Sigma-Aldrich), vortexed, spun down, snap-frozen on dry ice, and stored in -80C until further processing. A modified version of the SMART-Seq2 protocol (Picelli, et al. Nat Protoc. 2014.) was applied to generate full-length transcriptomic data from low-input samples—processed by the Klarman Cell Observatory and the Broad Institute Genomics Platform (GP). Briefly, FACS-purified cells were lysed in 5 μL of Buffer TCL (Qiagen) supplemented with 1% β-mercaptoethanol (Sigma-Aldrich) and snap-frozen until ready for processing. RNA from lysate was cleaned-up using Agencourt RNAClean XP SPRI magnetic beads (Beckman Coulter, A63987) and first strand cDNA synthesis was performed using Maxima H-minus Reverse Transcriptase (Thermo Fisher Scientific, EP0753) and amplified using KAPA HiFi HotStart PCR ReadyMix (KAPA Biosystems, KK2602). cDNA concentration was assessed using Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) and subsequently normalized to 0.25 ng μL-1. Library preparation was performed using the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096), confirmed to have a size of 500 bp using a BioAnalyzer (Agilent), and pooled libraries were normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flow cell cluster amplification and sequencing were performed according to the manufacturer's protocols using the paired-end Illumina sequencing (2 x 38 bp) with 75 cycle Nextseq 500 high output V2 kit (Illumina). Two rounds of sequencing were performed to generate sufficient read depth.