|
| Status |
Public on Jul 23, 2021 |
| Title |
het_CB3 |
| Sample type |
SRA |
| |
|
| Source name |
Soma of cultured dorsal root ganglia neurons
|
| Organism |
Mus musculus |
| Characteristics |
background strain: C57B6
genotype: GAR+/-
compartment: Soma
|
| Treatment protocol |
Adult total DRGs from 1 mouse were collected in Hanks’ buffered saline solution (HBSS) supplemented with 5uM Hepes, 10uM D-Glucose with antibiotics as per manufacturer instructions. DRGs were treated with 100 U of papain (P4762, Sigma) for 20 minutes, followed by treatment with 1 mg/ml collagenase-II (11179179001, Roche) and 1.2 mg/ml dispase-II (04942078001, Roche) for additional 25-30 minutes. DRGs were mechanically triturated in HBSS by aspiration in a glass Pasteur pipette whose opening was narrowed by fire polishing and was pre-covered in serum containing media. Cells were then laid on a 20% Percoll cushion (in L15 media) and recovered through centrifugation at 1000 g for 8 min. Cells were washed briefly in growth media (F12, 10 % Fetal Bovine Serum (FBS), Primocin) and plated on pre-coated Millicell 1m cell-culture inserts (MCRP06H48, Millipore) and allowed to grow for 3 days prior to extraction. Pre-coating included a first step of coating with Poly-L-Lysin (P4832, Sigma) followed by Laminin (23017-015, Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Qiagen RNeasy micro kit, following the manufacturer protocol. Briefly, samples were lysed in ± 300l RLT buffer supplemented with 400mM DTT by repeated vortexing (5 times, 4 seconds on low speed). Supernatants were kept and supplemented with 70% ethanol to reach 150% of recovered volume. Samples were loaded on columns and manufacturer protocol was followed. DNase treatment was conducted on-columns for 10 minutes. Elution of RNA from the columns was done using 13ul DNase/RNase free water.
TrueSeq Stranded RNA kit with RiboZero (Illumina )
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Basecalls/demultiplexing performed using Illumina CASAVA 1.8.2
Aligned to mm10 using STAR2.4.0
Count read using HT-seq0.6.1 using Ensembl GRCm38 as reference
Normalization (TMM), quantitation and differential expression analysis using EdgeR
Genome_build: mm10
|
| |
|
| Submission date |
Dec 16, 2019 |
| Last update date |
Jul 23, 2021 |
| Contact name |
Riki Kawaguchi |
| E-mail(s) |
rkawaguchi@mednet.ucla.edu
|
| Phone |
4244424783
|
| Organization name |
University of California Los Angeles
|
| Department |
Department of Neurology and Department of Psychiatry
|
| Lab |
Informatics Center for Neurogenetics and Neurogenomics (ICNN).
|
| Street address |
760 Westwood Plaza, Room 37-420
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095-1759 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE142096 |
Analysis of RNA localization in nucleolin deficient axons |
| GSE142576 |
GAR-Driven Subcellular Localization of the Growth Controlling RNA Binding Protein Nucleolin |
|
| Relations |
| BioSample |
SAMN13570996 |
| SRA |
SRX7387765 |
