|
| Status |
Public on May 05, 2016 |
| Title |
WT1.MDP.T2.5 |
| Sample type |
SRA |
| |
|
| Source name |
BMDM
|
| Organism |
Mus musculus |
| Characteristics |
tissue: bone marrow
cell type: macrophage
gender: male
strain: C57BL/6
age: 13 weeks old
plate: WT1
myd88 genotype: WT
replicate: 1
treatment: MDP
time: 2.5
|
| Treatment protocol |
4 hour time course in 0.5hr intervals with 12 dependent and independent innate immune ligands. RLT lysis in 200 uL and frozen at -80C.
|
| Growth protocol |
BMDM cultured from WT and Myd88 KO bones shipped o/n on wet ice from Al Shaw lab by Jean Wilson at Yale, gown in 30% FBS DMEM +P/S and 20% CMG, replaced fresh 30% Serum Media and 20% CMG on Day4 when transducing in 10cm dishes with 10x 58-MPRA pathway virus. Cells grown in 10 cm petri dishes until day 9 when they were scraped and counted on the hemocytometer and replated in 96-well format at a density of 125K cells per well.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RLT lysis, 50% SPRI purification, Turbo DNase, MaximaRT, SAv purification 25% (final 12.5% RNA lysate), Random hexamer 2nd strand cDNA, 2ng ds_cDNA tagmented for 12 minutes and run for 15 cycles PCR according to Nextera manufactures protocol
Modified SCRB-Seq, Soumillon, M., Cacchiarelli, D., Semrau, S., Oudenaarden, A. van & Mikkelsen, T. S. Characterization of directed differentiation by high-throughput single-cell RNA-Seq. bioRxiv 003236 (2014). doi:10.1101/003236; Modified Nextera protocol to PCR enrichonly the 3' end of mRNA transcripts and create digital gene expression libraries for Illumina sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Agencourt AMPURE XP Purified Total RNA
3 ng total from 12.5% lysate
|
| Data processing |
demultiplexing: The gene expression sequencing returns paired-end reads. Read 1 contains the plate-index, well-tag and RNA unique molecular identifier (UMI). Read 2 contains a fragment from the 3′ end of the transcript. The demultiplexing was done with a custom script that required perfect plate index and allowed one mismatch for the well tag.
mapping: The reads in read 2 were mapped to the refseq mouse mm10 gene using BWA with default parameters.
quantification: To quantify the expression genes we calculated the number of reads with unique UMI for each gene. The UMI information is embedded to the raw files as last 10 characters for every read description.
Genome_build: mm10
Supplementary_files_format_and_content: Processed data file contains counts of unique RNA molecules for every gene.
|
| |
|
| Submission date |
Nov 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Raivo Kolde |
| E-mail(s) |
rkolde@mgh.harvard.edu
|
| Organization name |
Massachusetts General Hospital
|
| Department |
Center for Computational and Integrative Biology
|
| Street address |
185 Cambridge St.
|
| City |
Boston |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
02114 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE75210 |
Simultaneous pathway activity inference and global gene expression analysis using RNA-sequencing [myd88] |
| GSE75212 |
Simultaneous pathway activity inference and global gene expression analysis using RNA-sequencing |
|
| Relations |
| BioSample |
SAMN04280054 |
| SRA |
SRX1439139 |
