|
| Status |
Public on Aug 19, 2013 |
| Title |
PITX1-RNAseq |
| Sample type |
SRA |
| |
|
| Source name |
Pitx1(wt) expressing chMM
|
| Organism |
Gallus gallus |
| Characteristics |
cell type: chicken micromass
days of incubation: 9
virus used for infection: RCASBP(A)-3xFLAG-cPITX1(wt)
chip antibody: none
|
| Growth protocol |
Chicken micromass cultures were routinely prepared by generating a single cell suspension of embryonic mesenchymal chicken limb bud cells (HH24) (2 × 10^7 cells/ml) and the appropriate virus was added before plating. Cultures were plated as 10µl droplets and cells were cultured for 9 days in chMM medium (DMEM/HAM’S F12 (Gibco) with 10% FBS (PAA), 0.2% chicken serum (Sigma), 1% L-glutamin (Gibco), 1% penicillin/streptomycin (Gibco) before harvest.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and protein-DNA complexes were enriched with antibody. For ChIP-seq library preparation ethanol precipitated DNA from ChIP was redissolved in 46 µl of water and subjected to library preparation using the NEBnext ChIP-Seq Library Prep Master Mix for Illumina (New England Biolabs) selecting for a fragment size range of 300-450 bp according to manufacturer's instructions. Sequencing was performed on an Illumina GAIIx sequencer with 36 bp single reads using TruSeq v5 sequencing chemistry and TruSeq v5 Cluster Generation Kits. For RNA-seq, total RNA from chicken micromass cultures was isolated using peqGOLD TriFast Reagent (peqLAB) and subsequent purification with RNeasy Mini Kit (Qiagen) according to manufacturer's instructions. mRNA for RNA-seq libraries was purified using the Oligotex mRNA Mini Kit (Qiagen) from 6 µg of total RNA. RNA-seq libraries were constructed using the NEBNext mRNA Library Prep Master Mix Set for Illumina according to manufacturer's instructions selecting for a fragment size range of 300-500 bp. Sequencing was performed on an Illumina GAIIx sequencer with 115 bp single reads using TruSeq v5 sequencing chemistry and TruSeq v5 Cluster Generation Kits.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
cDNA from polyA selected RNA
|
| Data processing |
The Reads Per Kilobase of transcript per Million mapped reads (RPKM) values were then calculated for all transcripts (i) in dependence on the formula introduced by Mortazavi et. al. Nat Methods 2008 : RPKM(i)= (reads(i) * 10^9)/(length(i) * no.of.reads), with reads(i) = number of reads mapped to the transcript; length(i) = length of this specific transcript ; no.of.reads = the number of mapped reads. The RPKM values were calculated for each isoform separately, taking into account the compensatory effect between isoforms of the same gene.
|
| |
|
| Submission date |
Mar 01, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Hansen |
| E-mail(s) |
peter.hansen@charite.de
|
| Phone |
+49 (0)30 450-569127
|
| Organization name |
Charité-Universitätsmedizin Berlin
|
| Department |
Institute for Medical Genetics and Human Genetics
|
| Lab |
Computational Biology Group
|
| Street address |
Augustenburger Platz 1
|
| City |
Berlin |
| ZIP/Postal code |
13353 Berlin |
| Country |
Germany |
| |
|
| Platform ID |
GPL13797 |
| Series (1) |
| GSE44799 |
Distinct Global Shifts in Genomic Binding Profiles of Limb Malformation Associated HOXD13 Mutations |
|
| Relations |
| SRA |
SRX248432 |
| BioSample |
SAMN01974730 |
