|
| Status |
Public on Nov 25, 2019 |
| Title |
naked mole-rat whole spleen saline control replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
whole spleen
|
| Organism |
Heterocephalus glaber |
| Characteristics |
tissue: whole spleen
condition: saline control
Sex: male
age: 23-25 months old
biological_replicate: 1
technical_replicate: NA
|
| Treatment protocol |
all animals were anasthesiasized with isoflurane. Saline controls received an intraperitoneal 200µl sterile saline injection. LPS-chllaneged animals received an intraperitoneal 1mg/kg lipopolysaccharide injection
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1ml TRIzol reagent (4C) (Invitrogen, 15596026) added to 20mg frozen splenic tissue in a 2ml microcentrifuge tube along with a single stainless-steel bead (Qiagen, 69965) and homogenization was performed using a Tissuelyser II (Qiagen) (30Hz for 2mins)
Illumina TruSeq Stranded Total RNA kit paired with the Ribo-Zero rRNA removal kit
Single cells were extracted using 10X Genomics Chromium Controller droplet emulsion capture. Whole tissue RNA was purified using 1ml TRIzol reagent (4C) (Invitrogen, 15596026) added to 20mg frozen splenic tissue in a 2ml microcentrifuge tube along with a single stainless-steel bead (Qiagen, 69965) and homogenization was performed using a Tissuelyser II (Qiagen) (30Hz for 2mins)
Single cell libraries were contructed using 10X Genomics Chromium Single-Cell 3’ Gel Bead and Library V2 Kit, suspending cells in PBS with 5% FBS to a final concentration of 1 x 10^6 cells/ml (1000 cells per ul) and loaded in each channel with a target output of 3000 cells. Reactions performed with Bio-Rad C100 Touch Thermal Cycler with a 96 Deep Well Reaction Module, 12 cycles for cDNA amplificationand OCR indexing. Whole spleen RNA libraries were contructed using Illumina TruSeq Stranded Total RNA kit paired with the Ribo-Zero rRNA removal kit
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
naked mole-rat whole spleen saline control replicate 1
|
| Data processing |
Single-cell fastq files were demultiplexed to their respective barcodes using the 10X Genomics Cell Ranger mkfastq utility
Unique Molecular Identifier (UMI) counts were generated for each barcode using the Cell Ranger count utility using the mm10 reference genome and the mouse Gencode version M12 annotation for mouse and HetGla2.0 reference genome along with the RefSeq GCF_000247695.1 annotation for naked mole-rat
For each single-cell sample barcodes that were not likely to represent captured cells were filtered out by detecting the first local minimum above 2 in a distribution of log10(#UMIs).
For each single-cell sample genes that are too sparsely captured across barcodes were filtered out by detecting the first local minimum above 3 in a distribution of log10(#barcodes)
For each single-cell sample barcodes capturing more than a single cell (multiplets) were sought as local modes in the distributions of log10(#genes) and log10(#UMIs), whose x-axis maxima are more than 1.5 higher than that of the x-axis location of the global maximum of the respective distribution and includes less than 5% of the total number of barcodes
Whole spleen RNA read data were mapped using STAR aligner version 2.5.3a
Transcript and gene abundances of whole spleen mapped RNA read data were quantified using MMSEQ
Genome_build: For mouse: mm10 reference genome and the mouse Gencode version M12 annotation. For naked mole-rat the HetGla2.0 reference genome and the RefSeq GCF_000247695.1 annotation for naked mole-rat
Supplementary_files_format_and_content: For the single-cell data processed data files are comma separated matrices of UMI counts where rows are gene IDs (Ensembl for mouse and Entrez for naked mole-rat) and columns are barcodes. The genes and barcode that are included are those that were retained following all filtering steps
|
| |
|
| Submission date |
Jun 13, 2019 |
| Last update date |
Nov 25, 2019 |
| Contact name |
Nimrod Daniel Rubinstein |
| E-mail(s) |
nrubinstein@calicolabs.com
|
| Phone |
9193082834
|
| Organization name |
Calico
|
| Street address |
1170 Veterans blvd
|
| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
| |
|
| Platform ID |
GPL26775 |
| Series (1) |
| GSE132642 |
Droplet-based single-cell RNA-sequencing of mouse and naked mole-rat spleen and circulating immune cells, in natural conditions, following lipopolysaccharide (LPS) challanege, and saline control |
|
| Relations |
| BioSample |
SAMN12046499 |
| SRA |
SRX6060847 |
