|
| Status |
Public on Jan 06, 2019 |
| Title |
mouse whole lung at PND1_Pool3 |
| Sample type |
SRA |
| |
|
| Source name |
mouse whole lung at PND1
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
age: PND1
genotype: wt
tissue: lung
|
| Treatment protocol |
The suspension was incubated in a 37°C water bath for 1 minute and then triturated for 30 seconds with cut tip 1 ml blue tip. The 1 minute 37°C incubation and 30 seconds trituration were repeated for a total of 7.5-10 minutes until most clumps were gone. Cell suspension was passed through a 40 micron strainer into 50 ml Falcon tube and the filter was rinsed with 5 ml of ice cold PBS with 0.1% BSA. Then 15 ml more were added to filtrate. Cells were transferred to 2X 14 ml conical blue tip tubes and spinned 300g for 5 minutes at 4°C. Supernatant was gently pipetted off. 5 ml of room temp Sigma RBC lysis buffer were added and lysis was monitored by removing a small aliquot and watching under a microscope. The suspension was incubated for 2-5 minutes at room temperature until red blood cells were lysed. 1/10 vol of 10X PBS was added, gently mixed, and spinned down 300g for 5 minutes at 4°C. Supernatant was discarded. Pellet was re-suspended in 0.8 ml of PBS with 0.1% BSA. Cells were settled on ice for about 2 minutes and then top 3/4 of cells were taken very gently. The settling onice and top 3/4 taking procedure was repeated once.
|
| Growth protocol |
Left and right lobes of PND1 mouse lungs were rapidly dissected in ice-cold PBS and then finely minced in a Petri dish on ice using a razor blade.Lung pieces were transferred to a 1.5 ml conical tubes using P1 pipetman, cut tip, using 700 ul of TrypLE(10X)/tube and then with 500 ul of collagenase (10mg/ml in PBS) per tube.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell concentration was examined with a hemocytometer and adjusted to around 300 cells per microliter for Fluidigm C1.Fluidigm C1 experiments were carried out as per Fluidigm recommended protocols.
Libraries were prepared using Illumina Nextera XT kit per illumina's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
mouse scRNAseq PND1 expression(TPM).xlsx
|
| 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.
paired reads were aligned against mm10 using tophat-Tophat 2.0.9.
Transcripts Per Kilobase Million(TPM ) values were computed using Partek (http://www.partek.com/) E/M quantification model.
Genome_build: mm10
Supplementary_files_format_and_content: Excel sheet including TPM expression for 130 cells
|
| |
|
| Submission date |
Nov 08, 2018 |
| Last update date |
Jan 06, 2019 |
| Contact name |
Yan Xu |
| E-mail(s) |
yan.xu@cchmc.org
|
| Phone |
513-6368921
|
| Organization name |
Cincinnati Children's Hospital Medical Center
|
| Street address |
3333 Burnet Ave
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE122330 |
Preparing for the first breath at single cell level [single cell Fluidigm C1] |
| GSE122332 |
Preparing for the first breath at single cell level |
|
| Relations |
| BioSample |
SAMN10396515 |
| SRA |
SRX4996515 |
