|
| Status |
Public on Nov 03, 2020 |
| Title |
AF1_49 |
| Sample type |
SRA |
| |
|
| Source name |
Sample 1B_embryonic thymus
|
| Organism |
Mus musculus |
| Characteristics |
mouse id: Sample 1B
strain: 5x polychromILC mice
tissue: Embryonic Thymus
|
| 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
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
SLX-16312.i701_i507.HWMJKBBXX.s_8.r
|
| Data processing |
Reads aligned to a modified mouse genome (addition of 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
|
| |
|
| Submission date |
Mar 10, 2020 |
| Last update date |
Nov 03, 2020 |
| Contact name |
Alastair Crisp |
| Organization name |
MRC Laboratory of Molecular Biology
|
| Street address |
Francis Crick Avenue
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0QH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE146740 |
Single cell RNA-seq (smartseq2) analysis of the ETP population from E15.5 embryonic thymus of the 5x polychromILC mice |
| GSE146745 |
RORa expression is a critical checkpoint for the bifurcation of the T cell and ILC2 lineages in the embryonic thymus |
|
| Relations |
| BioSample |
SAMN14348344 |
| SRA |
SRX7888566 |
