|Public on May 07, 2019
|primary spleen cells
cell type: primary spleen cells
molecule type: various proteins
|Spleen cells were obtained from C57/Blk6 mice, between 6-8 weeks of age. 16% formaldehyde solution (Thermo Scientific, 28908) was added directly to cells to a final concentration of 1.6% and incubated for 10 min at room temperature to fix the cells. 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
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.
Genome_build: 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.
|May 06, 2019
|Last update date
|May 09, 2019
|ROCHE SEQUENCING SOLUTIONS INC
|4300 Hacienda Drive
|Ultra-High Throughput Single Cell Analysis of Proteins and RNAs by Split-pool Synthesis