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| Status |
Public on Jan 16, 2024 |
| Title |
Colonic C3-expressing cells, scRNASeq |
| Sample type |
SRA |
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| Source name |
colon
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| Organism |
Mus musculus |
| Characteristics |
tissue: colon
genotype: C3-tdTomato reporter mouse line (C3IRES-tdTomato C57BL/6)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Colons from one male and one female C3IRES-tdTomato C57BL/6 mice were collected, and single cell suspensions were prepared. Live C3-expressing cells were sorted as DAPI-tdTomato+ using FACSAria (BD) into DMEM containing 10% FBS. A total of ~10,000 C3-expressing cells from each mouse were collected and pooled together.
Samples were loaded into Chip G per the user guide from 10x Genomics. The Chip G was then run on a 10x Chromium Controller. 100ul of GEM emulsion was taken from the chip, and inspected visually for inconsistencies in the recovered volume, the uniformity of the emulsion, or the relative amount of partitioning oil, and incubated at RT in a thermocycler (45 min at 53C, 5 min at 85C, then held at 4C until the next step). The emulsion was then broken by adding 125ul of Recovery Agent. The cDNA was then purified using Dynabeads MyOne SILANE. Per the 10x 3’v3.1 user guide, the 35ul of the purified cDNA was mixed with 15ul 10x cDNA primers (PN 200089) and 50ul of 10xAmp Mix (PN 2000047), then amplified on a thermocycler (98C for 3 minutes, [98C for 15 seconds, 63C for 20 seconds, 72C for 60 seconds], 12 repeats of the bracketed steps, 72C for 60 seconds, then held at 4 C. The amplified cDNA was then purified and separated based on size using a 0.6x Beckman Coulter SPRIselect Reagent cleanup. The larger bound fragments in the bead pellet containing transcript-derived cDNA were eluted in 35ul of Qiagen Buffer EB for use in Gene Expression library construction. Library size was measured by an Agilent Bioanalyzer 2100 High Sensitivity DNA assay and quantified using a Qubit dsDNA HS Assay kit on a Qubit 4.0 Fluorometer. A portion of the transcript-derived cDNA was fragmented, ligated to an Illumina Read 2 Sequencing Adaptor, and indexed with a unique 10x T Set A index, yielding the GEX library. 50ng of the transcript-derived cDNA was mixed with nuclease-free water up to a volume of 20ul, then mixed with 15ul of nuclease-free water, 5ul of 10x Fragmentation Buffer (PN 2000091), and 10ul of 10x Fragmentation Enzyme (PN 2000090), loaded onto a thermocycler, and incubated at 32C for 2 minutes, then 65 C for 35 minutes, and then held at 4 C. The fragmented product was purified with a 0.6x SPRI, with 75ul of the supernatant saved. The supernatant was purified with a 0.8x SPRI, with the pellet-bound DNA saved and eluted in 50uL. All 50ul of eluted sample was mixed with 20ul 10x Ligation Buffer (PN 2000092), 10ul DNA ligase (PN 220110), and 20ul of Adaptor Oligos (2000094), then incubated on a thermocycler at 20C for 15min. This product was purified using a 0.8x SPRI cleanup, with the pellet-bound DNA saved and eluted in 30ul. These 30ul were then mixed with 20ul of a unique 10X T Set A index and 50ul of 10x Amp Mix, then incubated on a thermocycler at 98 C for 45 seconds, [98C for 20 seconds, 54C for 30 seconds, 72C for 20 seconds], 13 repeats of the bracketed steps, 72C for 60 seconds, then held at 4C. The sample was then purified with a 0.6x SPRI, saving and transferring 150ul of the supernatant. The transferred supernatant was purified with a 0.8x SPRI and the pellet-bound DNA was eluted in 35ul. The finished library was measured on an Agilent BioAnalyzer and quantified on a Qubit 4.0. This library was then sequenced on an Illumina Novaseq SP 100, yielding approximately ~366M reads in the GEX.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw sequencing data in the form of binary files were converted into FASTQ files using the mkfastq command from CellRanger v6.1.0.
The FASTQ files were demultiplexed into gene expression libraries using CellRanger’s count command, and only reads mapping unambiguously and with less than 2 mismatches were kept
reads were mapped to the mouse mm10 transcriptome using CellRanger
Duplicate reads, those mapping to multiple regions or having a low alignment score (MAPQ < 10) were filtered out
A final gene expression matrix with genes in rows and cells in columns was then constructed
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Dec 15, 2023 |
| Last update date |
Jan 16, 2024 |
| Contact name |
CBDM Lab |
| E-mail(s) |
cbdm@hms.harvard.edu
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| Phone |
617-432-7747
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| Organization name |
Harvard Medical School
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| Department |
Microbiology and Immunobiology
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| Lab |
CBDM
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| Street address |
77 Avenue Louis Pasteur
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE250268 |
Gut complement induced by the microbiota combats pathogens and spares commensals |
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| Relations |
| BioSample |
SAMN38854243 |
| SRA |
SRX22897641 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7976452_barcodes.tsv.gz |
31.3 Kb |
(ftp)(http) |
TSV |
| GSM7976452_features.tsv.gz |
525.1 Kb |
(ftp)(http) |
TSV |
| GSM7976452_matrix.mtx.gz |
58.3 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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