Single-cell transcriptomes for paired uninflamed and inflamed mucosal tissues of CD patients
The single-cell transcriptomes were performed from paired uninflamed and inflamed mucosal biopsies obtained from surgically resected colonic samples from 3 CD patients. Overall design: The fresh colon tissues were stored in the sCelLiveTM Tissue Preservation Solution (Singleron) on ice after the surgery within 30 min. After digesting with sCelLiveTM Tissue Dissociation Solution (Singleron) and incubating with GEXSCOPE red blood cell lysis bu?er (RCLB, Singleron), the samples were stained with Trypan Blue and the cell viability was evaluated microscopically. Single-cell suspensions (2×10^5 cells/ mL) were loaded onto microwell chip using the Singleron Matrix Single Cell Processing System. Barcoding Beads were subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the Barcoding Beads and to obtain cDNA, and PCR amplification. The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE Single Cell RNA Library Kits (Singleron). Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads. Raw reads from scRNA-seq were processed to generate gene expression matrixes using CeleScope (https://github.com/singleron-RD/CeleScope) v1.5.2 pipeline. Briefly, raw reads were first processed with CeleScope to remove low quality reads with Cutadapt v1.17 to trim poly-A tail and adapter sequences. Cell barcode and UMI were extracted. UMI counts and gene counts of each cell were acquired with featureCounts v2.0.1 software, and used to generate expression matrix files for subsequent analysis. The 3 uninflammed samples are: DTJ-C, LWF-C, and TJY-C The 3 inflammed samples are: DTJ-D, LWF-D, and TJY-D