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| Status |
Public on May 11, 2019 |
| Title |
JW1_24 |
| Sample type |
SRA |
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| Source name |
Bone marrow
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| Organism |
Mus musculus |
| Characteristics |
cell phenotype from facs: Lin- Id2+ IL7ra+ Bcl11b+ CD25- Gata3lo/-
mouse id: 59146
tissue: Bone marrow
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| Extracted molecule |
total RNA |
| Extraction protocol |
Individual cells were flow cytometrically purified on a 96 well format into 0.2% Triton X-100 containing RNase inhibitor, dNTPs and oligo-dT primers and stored at -80oC.
On thawing, lysates were heated to 72 oC for 3 minutes and subject to reverse transcription, PCR preamplification (26 cycles) and PCR purification. cDNA library quality was assessed for all samples by qualitative PCR using primers for 18s RNA with an additional check by Bioanalyzer using an Agilent high sensitivity DNA chip on a small subset of libraries. A subset of libraries was quantified using the Qubit dsDNA HS assay kit and an average value used to calculate library dilution to 100-150 pg/ml. cDNA library tagmentation and amplification was performed using the Illumina Nextera XT DNA Library Preparation Kit according to manufacturer’s instructions except that all volumes were reduced 4-fold and tagmentation performed at 55oC for 20 minutes. Nextera index and Illumina adapter sequences were incorporated at the amplification stage (N7xx and S5xx). Amplified and indexed libraries were pooled and purified using Agencourt AMPure XP beads at a ratio of 1:0.9 library to beads and washed with 70% ethanol. Two rounds of purification were performed before a final elution in 1.25x total library volume of Nextera Resuspension buffer
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Single cell
ilc_R_sce_object.rds
ilc_single_cell_expression_matrix.tsv
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| Data processing |
Reads aligned to a modified mouse genome (addition of 92 ERCC spike-ins and the reporters found in the mouse) using the Salmon pseudoaligner (v0.8.2)
Pseudocounts analysis with the scater library (v1.6.3) (McCarthy et al., 2017), scran library (v1.6.9) (Lun et al., 2016), and sva library (sva: Surrogate Variable Analysis. R package v3.26.0).
Cells with pseudocounts below 3 median-absolute-deviations away from the median were removed. Genes with an average count across all remaining cells of less than 1 were removed.
Size factors were calculated with the scran library (based on the gene counts) and the data normalised by them.
Batch effects were removed using the ComBat empirical Bayes framework from the sva library
Genome_build: GRCm38.68
Supplementary_files_format_and_content: RDS of R SCE object containing metadata and gene expression generated using R,scater,scran and sva; tab-separated normalised gene expression from the SCE object
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| Submission date |
May 10, 2019 |
| Last update date |
May 12, 2019 |
| Contact name |
Alastair Crisp |
| Organization name |
MRC Laboratory of Molecular Biology
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| Street address |
Francis Crick Avenue
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0QH |
| Country |
United Kingdom |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE131038 |
Polychromic reporter mice reveal unappreciated innate lymphoid cell progenitor heterogeneity and elusive ILC3 progenitors in bone marrow |
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| Relations |
| BioSample |
SAMN11620045 |
| SRA |
SRX5819095 |
| 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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