|
| Status |
Public on Aug 01, 2014 |
| Title |
E11.5 MM Single Cell RNA-Seq-cell 47 |
| Sample type |
SRA |
| |
|
| Source name |
Analysis of single cells from E11.5 metanephric mesenchyme
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Tg(Crym-EGFP)82Gsat / MGI:4846969
component sampled: metanephric mesenchyme
theiler stage: TS19
age: 11.5 dpc
strain: mixed background
Sex: both
pooled sample: no
|
| Treatment protocol |
E11.5 kidneys of Crym-GFP mice were visualized with GFP fluorescence and microdissected to include the cells surrounding the ureteric bud and a few cells beyond the Crym boundary (Crym expression is the same as Six2 in the E11.5 kidney). Cells were rapidly dissociated with trypsin to single cell state, visualized under a microscope. The cells were adjusted to around 250/microliter and introduced into the Fluidigm C1 instrument for capture, wash, lysis and amplification with Clontech SMARTer technology according to Fludigm protocols. Samples were harvested, tagmented and barcoded, again according to Fluidigm protocols.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The single cell suspension was loaded into the Fluidigm C1 machine. The cell capture, wash and lysis were carried out according to standard Fluidigm protocols. Following capture individual capture sites were examined microscopically to record which chambers received single cells.
The reverse transcription and amplification reactions were carried out using standard Fluidigm (C1 Single-Cell Auto Prep System) methods with the SMARTer Ultra Low Input RNA for Illumina Sequencing Kit (Clontech) and the Advantage 2 PCR Kit (Clontech). The libraries were prepared with the Nextera XT DNA Sample Preparation Kit (Illumina Technologies). 1ng cDNA was suspended in Tagment DNA Buffer, and tagmentation (fragmentation and tagging with the adaptors) was performed with the Nextera enzyme (Tagment DNA Enzyme). Tagged and fragmented cDNA was purified with DNA Clean & Concentrator-5 (Zymo Research). The libraries were prepared by PCR with Nextera PCR Master Mix, PCR Primer Cocktail, 2 Nextera Indexes (Index 1 (i7) N701-N712 & Index 2(i5) N501-N508) and barcoded according to the following program: one cycle 98C for 30s, 5 cycles of 98C for 10s, 63C for 30s, 72C for 3min. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq2500 following the manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
http://www.gudmap.org/gudmap/pages/ngd_submission.html?id=GUDMAP%3A21744
|
| Data processing |
Processing step: Samples were aligned to the mm9 genome using TopHat v1.4.1. Known junctions from the RefSeq database were provided to TopHat with the -G option. The generation of RPKM data was carried out using GeneSpring 12.6.1-GX-NGS-RV (Agilent Technologies). Read density (nRPKM) in GeneSpring NGS is computed as the normalized read count (DESeq) per kilo base of exon model per Million reads. This is similar to the standard RPKM used in the literature except that it is computed on the normalized read count rather than on the raw read count.
Genome build: mm9
data file format and content: Excel spreadsheet including RPKM values for each Sample in Series.
|
| |
|
| Submission date |
Jul 07, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
GUDMAP Developers |
| E-mail(s) |
gudmap-db@gudmap.org
|
| Phone |
+44 131 651 8500
|
| Organization name |
IGMM MRC Human Genetics Unit
|
| Lab |
GUDMAP Database Group
|
| Street address |
Crewe Road
|
| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE59129 |
RNA-SEQ analysis of single cells extracted from the metanephric mesenchyme of E11.5 Crym-EGFP transgenic mice |
|
| Relations |
| BioSample |
SAMN02904277 |
| SRA |
SRX647611 |
