GSM4495743: sample 018-10 C.alb-spe T cells; Homo sapiens; RNA-Seq
First, CD154+ memory CD4 T cells were sorted on BD FACSAriaII, one cell per well, in 96-well plates containing specific lysis buffer at the CRTI, Nantes. Plates were immediately frozen for storage at -80°C, and sent on dry ice to the Genomics core facility of CIML, Marseille, for further generating scRNAseq libraries with the FB5P-seq protocol as described (Attaf et al., DOI: 10.3389/fimmu.2020.00216). Briefly, mRNA reverse transcription (RT), cDNA 5'-end barcoding and PCR amplification were performed with a template switching (TS) approach. After amplification, barcoded full-length cDNA from each well were pooled for purification and library preparation. For each plate, an Illumina sequencing library targeting the 5'-end of barcoded cDNA was prepared by a modified transposase-based method incorporating a plate-associated i7 barcode (Attaf et al., DOI: 10.3389/fimmu.2020.00216). Resulting libraries had a broad size distribution, resulting in gene template reads covering the 5'-end of transcripts from the 3rd to the 60th percentile of gene body length on average. As a consequence, sequencing reads cover the whole variable and a significant portion of the constant region of the TCRα and TCRβ expressed mRNAs, enabling assembly and reconstitution of TCR repertoire from scRNAseq data. Cells were analyzed in three distinct scRNAseq experiments (scRNAseqMetrics_SupplementaryTable_Geo.docx). Libraries prepared with the FB5P-seq protocol were sequenced on Illumina NextSeq550 platform with High Output 75 cycles flow cells, targeting 5x105 reads per cell in paired-end single-index mode with the following configuration: Read1 (gene template) 67 cycles, Read i7 (plate barcode) 8 cycles, Read2 (cell barcode and Unique Molecular Identifier) 16 cycles.
GEO
GSM4495743
GEO Accession
GSM4495743