GSM6027022: p6-3_F04; Mus musculus; RNA-Seq
Colonic epithelial cells were stained with eBioscience™ Fixable Viability Dye eFluor™ 506 (65-0866-14, Invitrogen) in a 1:1000 ratio for dead cell exclusion. Cells were Fc-blocked using Purified Rat Anti-Mouse CD16/CD32 (Clone 2.4G2, 553142, BD Pharmingen™) for 10 minutes and washed with FACS buffer (Ca2+ and Mg2+-free DPBS supplemented with 0.5% BSA and 0.3% sodium azide). For surface marker staining, PE Rat Anti-Mouse Siglec-F (Clone E50-2440, 552126, BD Pharmingen™) antibody and APC anti-mouse CD326 (EpCAM) antibody (clone G8.8, 118214, Biolegend) were used in a 1:50 and 1:100 dilution ratio, respectively. Antibody staining was performed for 30 minutes. All procedures were conducted at 4°C in the dark. Tuft cells were acquired by BD FACSAriaII (BD Biosciences) Single-cell RNA-Seq libraries were prepared using the Smart-seq2 protocol with few modifications. Single-cells were sorted using a BD FACSAriaII (BD Biosciences) into 96 well PCR plates (Thermo Scientific) containing 2μl of lysis buffer (0.1% Triton X-100, 1U/μl RNase Inhibitor (Enzynomics), 0.25μM oligo-dT30VN primer) and stored at -80°C. The plates were thawed on ice and incubated for 3 min at 72 °C to lyse the cells. Revers transcription (40U/µl Maxima H minus transcriptase), template switching reaction and PCR pre-amplification (15 cycles) were performed according to the protocol. The PCR products in each well was cleaned up using 0.5X SPRI beads (2% Sera-Mag Speed Beads (Cytiva), 1M NaCl, 10mM Tris-HCl pH 8.0, 1mM EDTA, 0.01% NP40, 0.05% Sodium Azide, 22% w/v PEG 8000). To exclude empty wells and low-quality libraries, cDNA libraries were assessed by quantitative PCR with a primer pair of GAPDH, a housekeeping gene (forward: 5′-GTCGTGGAGTCTACTGGTGTCTTCAC-3′; reverse: 5′-GTTGTCATATTTCTCGTGGTTCACACCC-3′). Samples showing a high threshold cycle (Ct) value (>35 cycles) or no PCR amplification were discarded. 50–200 pg of each cDNA library were used to generate the Illumina sequencing library using EZ-Tera XT DNA library preparation kits (Enzynomics). After the final PCR amplification, samples were pooled and size-selected by SPRI beads (removing DNA bound to 0.3X beads and taking DNA bound to subsequent 0.6X beads). Pooled sequencing libraries were sequenced on an Illumina NextSeq 500 instrument using a 38-bp paired-end reads setting.