|
| Status |
Public on Aug 17, 2012 |
| Title |
HEK293 cells transfected with a 1:1 mixture of pBluescript and pEGFP-C1 plasmids |
| Sample type |
SRA |
| |
|
| Source name |
cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
passages: 5-15
transfected plasmid: pBluescript + pEGFP-C1
|
| Treatment protocol |
HEK293 cells were transiently transfected with 3 micrograms of phRL-SV40, pGL4-SV40 or a 1:1 mixure of pBLuescript and pEGFP-C1 plasmids (per well in a 6-well plate) using TurboFect (Fermentas) transfection reagent.
|
| Growth protocol |
HEK293 cells were grown under standard tissue culture conditions (DMEM + 10% FCS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNAzol RT (Molecular Research Center). Further sample processing and SOLiD sequencing was performed by Seqomics Ltd. (Hungary). Briefly, samples were depleted of ribosomal RNA using RiboMinus rRNA Removal Kit, eluted twice into 15 ul and treated with DNase I (5 ul 10x DNase I buffer, 2.5 U DNase I and water up to 50 ul reaction) 20 min at 37°C. Samples were ethanol-precipitated, eluted into 10 ul TE buffer and fragmented by RNase III treatment (1 ul 10x RNaseIII buffer, 8 ul sample, 1 ul RNaseIII) 10 min at 37°C. Fragmented samples were precipitated by ethanol and eluted into 4 ul. Adaptor was hybridized and ligated to each sample (10 ul 2x ligase buffer, 2 ul Adaptor mix, 3 ul hybridization solution, 2 ul ligation enzyme) by incubating O.N. at 16°C. Adaptor-ligated RNA was subjected to reverse transcription (4 ul 10x RT buffer, 2 ul dNTP mix, 2 ul RT primer, 1 ul ArrayScript RT, 13 ul water) for 40 min at 42°C. Resulting cDNA was purified using PCR Purification kit (Qiagen) and eluted into 10 ul. cDNA was run on 6% TBE-Urea gel and fragments 150-250 nt in length were excised. Isolated cDNA was amplified for 15 cycles using AmpliTaq DNA polymerase and purified by PCR Purification kit (elution twice in 10 ul). Second round of size selection on PAGE gel was performed and PCR products 110-130 nt in length were excised from gel and isolated. Purified amplicons were sequenced using Abi SOLiD 4 platform.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
AB SOLiD 4 System |
| |
|
| Description |
pBS-EGFP
pBluescript GenBank accession number: U26267.1
pEGFP-C1 GenBank accession number: U55763.1
When we looked for the plasmid sequence, we relied on the manufacturer's websites and datasheets (links will be included in the paper), rather than on the GenBank.
|
| Data processing |
Sequence reads of 18-50 nt length (after subtracking the adaptor sequence) were obtained and mapped onto specified plasmid allowing only perfectly matched reads. In the second round of mapping (Edited reads), A-to-G mischatches were allowed during mapping.
Columns in processed files are as follows: Sequence = sequence of the particular read mapped; Count = number indicating, how many times is the particular read present in the dataset; Strand = indicates if the read was mapped on a sense or an antisense strand of the plasmid; Length = length of the read; Edited nucleotides = number of A-to-G mismatches present within the mapped read.
|
| |
|
| Submission date |
Feb 24, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Radek Malik |
| E-mail(s) |
malikr@img.cas.cz
|
| Organization name |
Institute of Molecular Genetics of the ASCR
|
| Department |
Dep. of Epigenetics Regulations
|
| Street address |
Videnska 1083
|
| City |
Prague 4 |
| ZIP/Postal code |
CZ-14220 |
| Country |
Czech Republic |
| |
|
| Platform ID |
GPL13393 |
| Series (1) |
| GSE36062 |
Complexity of expression from transiently transfected plasmids |
|
| Relations |
| SRA |
SRX122236 |
| BioSample |
SAMN00792330 |
