|
Status |
Public on Jul 07, 2016 |
Title |
MN_SCR_KD_Rep2 |
Sample type |
SRA |
|
|
Source name |
SCR knockdown in MN
|
Organism |
Homo sapiens |
Characteristics |
cell line: H3 ES cells ectopic expression construct: GFP under 9kb murine Mnx1 promoter cell treatment: siRNA knockdown by nucleofection + 72 hrs in neural culture media + Retinoic Acid
|
Treatment protocol |
nucleofection of 20 pmol siRNA
|
Growth protocol |
human ES in mTesR1 differentiated by media change: grown in 1:1 DMEM/F12 (Life Technologies) and NeuroBasal (Life Technologies), 2 mM glutamax-I (Life Technologies), 1x N2 supplement (Life Technologies), 1x B27 supplement (Life Technologies) 10nM SB431542 (Sigma), 1 μM dorsomorphin (Millipore) and 0.1 μM retinoic acid (RA) (Sigma). Media changed daily
|
Extracted molecule |
total RNA |
Extraction protocol |
Trizol, cleanup/DNAse with Zymo clean and concentrate, illumina Truseq to make cDNA from cDNA or sheared DNA: blunt ends, A-tail, ligate illumina sequencing adapters, PCR with full length/indexed illumina primers
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
|
|
Description |
knockdown of control (scramble)
|
Data processing |
Basecalls performed using OLB 1.8 RNA-Sequences were aligned to the hg9 transcriptome using tophat 2.4 EdgeR was uset to compute RPKM values DNA-Sequences were aligned to the hg19 genome using bwa 0.5.9 For one batch of libraries, ("rep2" RNAseq and ChIP samples) high contamination of a single probe made in parallel for another experiment (chr7:15630900-156311000) was observed and the spike was removed in ChIP files Aligned and filtered sequences were used to compute input normalized read densities using the SPP software (bw files) Genome_build: hg19
|
|
|
Submission date |
Sep 18, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Robert E Kingston |
E-mail(s) |
kingston@molbio.mgh.harvard.edu
|
Organization name |
MGH/Harvard
|
Department |
Molbio
|
Lab |
Kingston
|
Street address |
185 Cambridge Street
|
City |
Boston |
State/province |
Massachusetts |
ZIP/Postal code |
02114 |
Country |
USA |
|
|
Platform ID |
GPL11154 |
Series (1) |
GSE61549 |
A long non-coding RNA tunes PRC1 function during human and zebrafish development |
|
Relations |
BioSample |
SAMN03074037 |
SRA |
SRX706337 |