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Sample GSM2171871

Query DataSets for GSM2171871

Status

Public on May 21, 2016

Title

CTRL, rep2

Sample type

SRA

Source name

E18.5 mouse embryo

Organism

Mus musculus

Characteristics

cell type: Ureteric epithelial cells
tissue: Kidney
age: 18.5 days
genotype: Esrp1 +/+, Esrp2 -/-

Extracted molecule

total RNA

Extraction protocol

E18.5 embryonic kidney pairs isolated from individual embryos were placed in PBS on ice, then transferred into 500 μl of 0.03% collagenase (Sigma, Cat#C1889) in PBS for 10 minutes at 37ºC. Samples were transferred to ice and the kidneys were dissociated by trituration using an 18 gauge needle with 8-10 passes followed by a 25 gauge needle for 8-10 passes to homogenize to a single-cell slurry. Cells were transferred to a 15ml falcon tube containing 4-5mls 2% fetal bovine serum (FBS) in PBS to inhibit collagenase. The cells were pelleted for 5 minutes at 400xg (4ºC) and washed three times in 5 mls of 2% fetal bovine serum (FBS). The final cell pellet was resuspended in 60ul 2% FBS in PBS and transferred to an Eppendorf tube. Cell count and viability was measured using 5 μl of cells + 5 μl trypan blue and a Hemocytometer. 5 μl of the remaining cell volume from each sample was pooled to serve as an unstained control. The remaining 50ul was processed for labelled with FITC-DBA lectin. The cells were pelleted at 400xg for 5 min. and stained in a fresh 50 μl of staining solution on ice and in the dark with DBA-FITC (Vector Labs, Cat#FL-1031) 1:10 diluted in 2% FBS in PBS for 20min. The cells were washed in 1mL of 2% FBS in PBS and centrifuged at 400 x g (4ºC) for 5min. The cell pellet was resuspended in 300-400 μl of 2% FBS in PBS for FACS. GFP positive gated cells were isolated using the unstained cells as a negative control. GFP+ cells were collected directly into 500 μl of Trizol (ThermoFisher) and snap frozen on dry ice and stored at -80ºC.
RNA libraries were prepared for sequencing using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (mRNA) (New England Biolabs)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

CTRL

Data processing

Illumina Casava1.7 software used for basecalling.
Sequenced reads were mapped to Ensembl transcript (release 72) then mm10 whole genome using tophat v1.4.1 with parameters -a 8 -m 0 -I 300000 -p 8 -g 20 --library-type fr-unstranded --initial-read-mismatches 3 --segment-mismatches 2
Fragments Per Kilobase of exon per Million fragments mapped (FPKM) were calculated using cuffdiff v2.2.1 with uniquely and properly mapped pairs.
Genome_build: mm10
Supplementary_files_format_and_content: an excel file includes FPKM values for each Sample

Submission date

May 20, 2016

Last update date

May 15, 2019

Contact name

Yi Xing

Organization name

UCLA

Department

Department of Microbiology, Immunology, & Molecular Genetics

Street address

650 Charles E. Young Dr. South