normalization data transformation protocol
Use of cellranger count as recommanded by 10X Genomics (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
high throughput sequence alignment protocol
Bioinformatics analysis was performed using 10X Genomics recommendations. Briefly, fastq files were created with cellranger mkfastq, and cellranger count was used to perform alignment, filtering, barcode counting, and unique molecular identifier (UMI) counting.
nucleic acid sequencing protocol
6,000 cells with a viability of 94% were processed to finally sequence 3,625 of them. Sequencing was done on Illumina HiSeq 4000 sequencer.
sample collection protocol
One cryotube (1.7 million cells) of the M398 healthy cell line was thawed in a T75 BioCoat™ Collagen I cultureware (356485, Corning Incorporated) with SKM02 medium (AMSbio). Cells were harvested with trypsin the following day. 1.5 million cells were centrifuged at 300 g for 5 minutes. Cells were resuspended at 1,500 cells/µL in DMEM F-12 with 10% serum and processed according to the 10X Genomics single-cell RNA sequencing protocol.
nucleic acid extraction protocol
nucleic acid library construction protocol
The library was prepared with Chromium™ Single Cell 3' Library & Gel Bead Kit v2.