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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 08, 2014 |
| Title |
E14.5, biological replicate 1, single cells, transcriptome 23 |
| Sample type |
SRA |
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| Source name |
distal lung epithelium
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
tissue: lung
age: Embryonic day 14.5
genotype: wild type
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| Treatment protocol |
To obtain AT2 cells from the adult lung, an adult mouse whose genome contained a tetracycline-responsive, nuclear reporter only transcribed in cells transcribing Sftpc (Sftpc-Cre-ERT2-rtta-/- tetO-HIST1H2BJ-GFP+/-) was injected with 2 mg of doxycycline (Sigma) and used 3 days afterwards. Pregnant and non-pregnant adult mice were euthanized by CO2 administration. For embryonic samples (E14.5, 16.5, and 18.5), embryonic lungs were isolated and either a single embryonic lung was used or 5-7 sibling lungs were pooled. For the adult experiment, the pulmonary vasculature was cleared and lungs were gently inflated with digestion buffer prior to isolation and mincing of the lung. All tissue was digested at 37C for 45 min using Elastase and DNase I, triturated and subsequently filtered using 100µm and 40µm strainers to obrtain a single cell suspension. Embryonic lung epithelial cells were purified by magnetic-assisted cell sorting using CD45 depletion followed by EpCAM enrichment. Transgenically labeled adult AT2 cells were purified by fluorescence-assisted cell sorting.
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| Growth protocol |
Mice were housed in filtered cages and all experiments were performed in accordance with approved IACUC protocols. Timed-pregnant C57BL/6J females (JAX) were used for all embryonic time points reported with gestation age verified by crown-rump length prior to use. For adult mice, a transgenic-labeling approach was employed. Mice were bred to be homozygous for a knock-in allele into Sftpc encoding for a reverse tetracycline transactivator (SftpC-Cre-ERT2-rtTA) and heterozygous for an inserted transgene, which drives the expression of a GFP tagged human histone 1 in a tetracycline-dependent manner (tetO-HIST1H2BJ/GFP).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were loaded into the Fluidigm C1 microfluidic platform, where single cells were captured. Lysis of single cells, reverse-transcription of mRNA into cDNA as well as preamplification of cDNA occured within the microfluidic device using reagents provided by Fluidigm as well as the SMARTer Ultra Low RNA kit for Illumina (Clontech). External RNA spike-in transcripts (ERCC spike-in Mix, Ambion) were added to all single cell lysis reactions. As a bulk control, cDNA was prepared from approximately 200 cells in a PCR tube using the same protocol and reagents as used for the single cells. In addition, one "no cell control" was prepared using reagents without any cell input in a PCR tube.
Libraries were prepared using Illumina Nextera XT kit per illumina's protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
E14.5, biological replicate 1, single cells
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| Data processing |
CASAVA 1.8.2 was used to separate out the data for each single cell using unique barcode combinations from the Nextera XT preparation and to generate *.fastq files.
adapter sequences were removed using cutadapt-1.2.1
trimming by base quality was performed and low complexity reads were removed using prinseq-lite-0.20.3
paired reads were aligned against mm10 using bowtie2-2.1.0 and tophat-2.0.8
Fragments Per Kilobase of transcript Per Million mapped reads (FPKM) values were computed using cufflinks-2.0.2
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample.
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| Submission date |
Nov 20, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Barbara Treutlein |
| E-mail(s) |
[email protected]
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| Organization name |
Stanford University
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| Department |
Bioengineering
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| Lab |
Quake
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| Street address |
318 Campus Drive
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| City |
Stanford CA |
| State/province |
CA |
| ZIP/Postal code |
94305-5432 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE52583 |
High throughput quantitative whole transcriptome analysis of distal mouse lung epithelial cells from various developmental stages (E14.5, E16.5, E18.5 and adult) |
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| Relations |
| BioSample |
SAMN02415528 |
| SRA |
SRX380549 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1271967_E14_1_C65_IL3938.sorted.genes.fpkm_tracking.gz |
644.7 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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