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GEO help: Mouse over screen elements for information. |
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Status |
Public on Oct 25, 2020 |
Title |
P2278_N707_S502 |
Sample type |
SRA |
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Source name |
Thymocytes
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Organism |
Mus musculus |
Characteristics |
replicate: replicate_1 genotype: WT cell type: Thymocytes strain: C57BL/6 cell population: TCRhiDP protocol: Smart-Seq2
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Extracted molecule |
total RNA |
Extraction protocol |
For replicate 1, full-length single-cell RNA-seq libraries were prepared using the Smart-seq2 protocol (Picelli et al., 2013) with minor modifications. Thymocytes were sorted into 96-well plates containing the lysis buffer (0.2% Triton-100, 1U/µl RNase inhibitor)Reverse transcription was performed using SuperScript II (ThermoFisher Scientific) in the presence of 1 μM oligo-dT30VN (IDT), 1 μM template-switching oligonucleotides (QIAGEN), and 1 M betaine. cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Kapa Biosystems) and IS PCR primer (IDT), with 25 cycles of amplification. For replicate 2, full-length single-cell RNA-seq libraries were prepared using the SMART-Seq v5 Ultra Low Input RNA Kit for Sequencing (Takara Bio). All reactions were downscaled to one quarter of the original protocol and performed following thermal cycling manufacturer’s conditions. Thymocytes were sorted into 96-well plates containing 2.5 µl of the Reaction buffer (1x Lysis Buffer, RNase Inhibitor 1U/µl). Reverse transcription was performed using 2.5 µl of the RT MasterMix (SMART-Seq v5 Ultra Low Input RNA Kit for Sequencing, Takara Bio). cDNA was amplified using 8 µl of the PCR MasterMix (SMART-Seq v5 Ultra Low Input RNA Kit for Sequencing, Takara Bio) with 25 cycles of amplification. Replicate 1 followed purification of amplified cDNA with Agencourt Ampure XP beads (Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). A total of 140 pg of the amplified cDNA was fragmented using Nextera XT (Illumina) and amplified with Nextera XT indexes (Illumina). Products of each well of the 96-well plate were pooled and purified twice with Agencourt Ampure XP beads (Beckmann Coulter). Final libraries were quantified and checked for fragment size distribution using a Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled sequencing of Nextera libraries was carried out using a HiSeq2000 (Illumina) to an average sequencing depth of 0.5 million reads per cell. Sequencing was carried out as paired-end (PE75) reads with library indexes corresponding to cell barcodes. Replicate 2 followed purification of amplified cDNA with Agencourt Ampure XP beads (Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). A total of 140 pg of the amplified cDNA was fragmented using Nextera XT (Illumina) and amplified with double indexed Nextera PCR primers (IDT). Products of each well of the 96-well plate were pooled and purified twice with Agencourt Ampure XP beads (Beckmann Coulter). Final libraries were quantified and checked for fragment size distribution using a Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled sequencing of Nextera libraries was carried out using a HiSeq4000 (Illumina) to an average sequencing depth of 0.5 million reads per cell. Sequencing was carried out as paired-end (PE100) reads with library indexes corresponding to cell barcodes.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Data processing |
Smart-seq2 and SMARTer sequencing data were aligned with TopHat2 version 2.1.1 (Kim et al., 2013) GRCm38 (mm10) mouse genome reference was used for alignment and gene annotation from UCSC. Read counts for genes were calculated using velocyto version 0.17 (La Manno et al., 2018) and the cell-gene count matrix obtained was used for downstream analysis. Genome_build: GRCm38 (mm10) Supplementary_files_format_and_content: CSV file containing the raw read counts matrix of all the samples from the experiment calculated using the tool velocyto.
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Submission date |
Apr 23, 2020 |
Last update date |
Oct 25, 2020 |
Contact name |
LMS Bioinformatics Core |
E-mail(s) |
bioinformatics@lms.mrc.ac.uk
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Organization name |
MRC London Institute of Medical Sciences
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Department |
Bioinformatics Core
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Street address |
Hammersmith Hospital Campus, Du Cane Road
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City |
London |
ZIP/Postal code |
W12 0NN |
Country |
United Kingdom |
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Platform ID |
GPL13112 |
Series (1) |
GSE149207 |
scRNA-seq of mouse thymocyte CD4 CD8 lineage choice and differentiation |
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Relations |
BioSample |
SAMN14678708 |
SRA |
SRX8164497 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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