|Public on Nov 27, 2019
|PBMC primary cells from donor
|cell type: PBMC primary cells from donor
molecule type: various proteins
|Cells at a density of ~1×10^6/mL were fixed in RPMI medium without serum in 1.6% paraformaldehyde (Electronic Microscopy Sciences) for 10 min at room temperature under gentle agitation. Cells to be analyzed for only protein expression were stored in 1X PBS with 10% DMSO at -80°C until antibody staining.
|QIAquick Gel Extraction Kit (Qiagen, 28704).
PCR with Illumina dual index primers
|Library strategy: Single cell barcoding by split-pool synthesis via ligation
FLASH-1.2.11 was used to merge the paired end reads.
seqtk-v1.2 was used to convert merged fastq file to fasta format.
The reads were then parsed to identify cell-id and marker-id based on the white-list info in the associated qdata folder, deduplicated based on cell-id and marker-ids and finally chimeric reads were filtered out.
Expression counts were then used to remove putative debris and doublets cells based on the criteria listed in Singlet_settings.txt in the corresponding qdata folder.
Expression counts matrix was then normalized by scaling the counts by row-means and multiplying by a factor of 10.
A github repository for the software used to process the raw-fastqs is available at the following location: https://github.com/bioinform/QBC_Single_Cell_Analysis_NGS
Supplementary_files_format_and_content: Tab separated text files containing jittered expression counts (both normalized and un-normalized) for different markers for each individual cell
The *tar.gz contains various files containing white-list information for cell-barcode ids, anchor-sequence info, marker-sequence info and settings information for removal of cell-debris and cell-doublets. The files in this folder is used to process the raw fastqs for each of the experiment.
|Nov 26, 2019
|Last update date
|Nov 27, 2019
|ROCHE SEQUENCING SOLUTIONS INC
|4300 Hacienda Drive
|Ultra-High Throughput Single Cell Analysis of Proteins and RNAs by Split-pool Synthesis