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| Status |
Public on Jul 25, 2022 |
| Title |
KO3_5 |
| Sample type |
SRA |
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| Source name |
dermal mast cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J Mcpt5-Cre Tln1fl/fl R26LSLYFP
tissue: Ear skin
reporter/marker: Mcpt5 Cre+/-
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| Treatment protocol |
no treatment
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| Growth protocol |
Mast cells were isolated from ear skin of C57BL/6J wt and Tln knockout mice.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mast cells were isolated from ear skin of C57BL/6J Mcpt5-Cre wt and Tln knockout mice .
The amplified RNA (aRNA) was reversely transcribed in order to generate libraries for sequencing. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
Library of 92 cells
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| Data processing |
For image aquisition, intensity extraction and basecalling HiSeq Control Software 2.0.2, RTA 2.4.11 / Recipe Fragment 2.0.0.2 was used. Conversion of bcl2fastq files was performed using bcl2fastq 2.17.1.14
Paired end reads were aligned to the transcriptome using bwa (version 0.6.2-r126) with default parameters. The transcriptome contained all RefSeq gene models based on the mouse genome release mm10 downloaded from the UCSC genome browser. All isoforms of the same gene were merged to a single gene locus.
The right mate of each read pair was mapped to the ensemble of all gene loci and to the set of 92 ERCC spike-ins in sense direction (Baker et al., 2005). Reads mapping to multiple loci were discarded.
The left read contains the barcode information: the first six bases corresponded to the cell specific barcode followed by six bases representing the unique molecular identifier (UMI). The remainder of the left read contains a polyT stretch. The left read was not used for mapping to the reference transcriptome.
For each cell barcode, the number of UMIs per transcript were counted and aggregated across all transcripts derived from the same gene locus.
Based on binomial statistics, the number of observed UMIs were converted into transcript counts (Grün et al., 2014).
Assembly: mm10
Supplementary files format and content: CSV files, columns represent each cell barcode (total barcodes used = 192), rows represent the geneid and the values in the file are the quantified number of transcripts.
Supplementary files format and content: cellbarcodes_cellid.csv is a supplemetary file contains cellds and one of the 192 unique cellbarcode associated with the cell id.
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| Submission date |
Jun 02, 2022 |
| Last update date |
Jul 25, 2022 |
| Contact name |
Nadim Aizarani |
| Organization name |
FMI
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL21493 |
| Series (1) |
| GSE205412 |
Slow-integrin dependent migration organizes networks of tissue-resident mast cells |
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| Relations |
| BioSample |
SAMN28852701 |
| SRA |
SRX15577305 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6211611_KO_3_5.coutt.csv.gz |
414.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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