This dataset is part of the Alternative transcript isoform detection with single cell and spatial resolution Application Note. The Chromium library input files, Cell Ranger output files, and Oxford Nanopore library FASTQ files are provided in the Inputs tab.
Cryopreserved, dissociated tumor cells from Stage III squamous cell lung carcinoma (lung cancer DTCs) were obtained from Discovery Life Sciences. Cells were prepared as previously described (CG000233).
5' Single Cell Gene Expression Library
Libraries were generated from ~4,000 cells (2,616 cells recovered) as described in the Chromium Single Cell 5' Reagent Kits User Guide (v2 Chemistry Dual Index) (CG000331 Rev E) using the Chromium X and sequenced on an Illumina NovaSeq 6000 to a read depth of approximately 25,000 mean reads per cell.
Paired-end, dual indexing:
- Read 1: 28 cycles (16 bp barcode, 10 bp UMI)
- i5 index: 10 cycles (sample index)
- i7 index: 10 cycles (sample index)
- Read 2: 90 cycles (transcript)
Oxford Nanopore Technologies library
Oxford Nanopore cDNA libraries were prepared starting from full-length, 10x barcoded cDNA using the PCR-cDNA Sequencing Kit per the manufacturer’s protocol, "Single-cell transcriptomics with cDNA prepared using 10X Genomics".
Each long-read sequencing library was loaded onto an individual PromethION Flow Cell. Libraries were sequenced for 72 hours and base calling was performed using MinKNOW’s Super Accurate Base Calling mode (MinKNOW software version 22.03.04).