GSM8676182: INT_P6_RNA_sequencing; Homo sapiens; RNA-Seq
Intestinal biopsy were obtained via colonoscopy. Intestinal biopsy samples were processed for scRNA-seq. Peripheral blood samples were obtained and processed for scRNA-seq.Samples were immediately subjected to single-cell dissociation within 3 hours after collection. Briefly, the tissue samples were first washed by Hanks Balanced Salt Solution without Ca2+,Mg2+(D-HBSS) to remove blood and fecal residues, then enzymatically dissociated in digestion medium (1mg/ml Collagenase D,100ug/ml DNAse Ⅰ in D-HBSS) for 1 hour. The digestion was stopped by adding 10 ml washing buffer (2mM EDTA,2%FBS in D-HBSS). The cell suspension was filtered using a 30um nylon mesh. Peripheral blood mononuclear cells (PBMCs) from Peripheral blood samples were isolated using density gradient centrifugation through lymphocyte separation medium. CD8+T cells from intestinal and PBMC suspensions were isolated by magnetic bead separation (human CD8 MicroBeads kit, Miltenyi Biotec) according to the manufacturer's instructions. The numbers of CD8+T cells were determined using an automatic cell counter. Single-cell RNA-seq and V(D)J-seq libraries were prepared using the SeekOne® DD Single Cell 5' Library Preparation Kit (SeekGene Catalog No. K00501) and the SeekOne® DD Single Cell V(D)J Enrichment Kit (SeekGene Catalog No. K00601). Briefly, an appropriate number of cells were mixed with the reverse transcription reagent and loaded into the sample well of the SeekOne® DD Chip S3. Gel Beads and Partitioning Oil were then dispensed into corresponding wells in the chip. After emulsion droplet generation, reverse transcription was performed at 42°C for 90 minutes, followed by inactivation at 85°C for 5 minutes. Next, cDNA was purified from broken droplets and amplified via PCR. For the RNA-seq library, the amplified cDNA was fragmented, end-repaired, A-tailed, and ligated to sequencing adapters. In the case of V(D)J-seq, the amplified cDNA from the 5' cDNA was enriched for V(D)J regions. Both libraries underwent indexed PCR to amplify the DNA representing the 3' polyA region of expressed genes, which also contained the Cell Barcode and Unique Molecular Index (UMI). The indexed libraries were cleaned up with VAHTS DNA Clean Beads (Vazyme N411-03), analyzed using Qubit (Thermo Fisher Scientific Q33226) and Bio-Fragment Analyzer (Bioptic Qsep400). Finally, the libraries were sequenced on the Illumina NovaSeq 6000 with a PE150 read length.