|
| Status |
Public on Jan 26, 2019 |
| Title |
Crush control replicate 3A |
| Sample type |
SRA |
| |
|
| Source name |
Crush control
|
| Organism |
Mus musculus |
| Characteristics |
strain background: mixed: C57BL/6 and AJ
age: 8-9 weeks
Sex: female
genotype: Nav1.8-Cre; Tsc2 flox/+; Rosa26-ZsGreen
tissue: L4 dorsal root ganglia ipsilateral to sciatic nerve crush
cell type: Nav1.8-positive dorsal root ganglia neurons
sorted fraction: 100 FACS-sorted GFP-positive cells
|
| Treatment protocol |
Sciatic nerve of mice was crushed with forceps.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
3 days post-injury mice were sacrificed. L4 dorsal root ganglia contralateral to injury were isolated, dissociated with papain and collagenase and passed through 70 um filter. Cells were FACS sorted for GFP-positive cells into Clontech lysis buffer with 5% RiboLock Rnase Inhibitor (Thermo Fisher).
Library preparation was performed with ~100 cells collected by flow cytometry in 10 ul of Clontech’s reaction buffer using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Clontech) per manufacturer’s protocol. Three technical replicates of 100 cells each (A-C) were obtained for each biological replicate (1-4). cDNA was amplified for 13 cycles. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty cycle 20%, cycles/burst 50, time 120 seconds at room temperature. cDNA was blunt ended, had an A base added to the 3’ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 15 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina HiSeq-3000 using single reads extending 50 bases.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
crush_control_3A
|
| Data processing |
Samples were QC’d using FastQC and not trimmed
Reads were aligned to mm10 using STAR-align with default settings Reads aligned to mm10 using STAR-align with default settings
Reads aligned to genes were counted using Htseq-counts with default settings
Technical replicates were collapsed in DESeq2
Differential expression was calculated using DESeq2
Genome_build: mm10
Supplementary_files_format_and_content: Counts files generated from HTseq-count
|
| |
|
| Submission date |
Jan 25, 2019 |
| Last update date |
Jan 26, 2019 |
| Contact name |
Dan Carlin |
| E-mail(s) |
carlind@wustl.edu
|
| Phone |
314-362-0155
|
| Organization name |
Washington University in St Louis
|
| Department |
Neuroscience
|
| Lab |
Valeria Cavalli
|
| Street address |
660 S Euclid Ave / Campus Box 8108
|
| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL21493 |
| Series (1) |
| GSE125685 |
RNA-seq analysis of FACS-sorted control and Tsc2 cKO nociceptors with and without sciatic nerve crush |
|
| Relations |
| BioSample |
SAMN10821712 |
| SRA |
SRX5293799 |
