|
| Status |
Public on Jun 21, 2018 |
| Title |
AcBac_AAV2wt_48h_2 |
| Sample type |
SRA |
| |
|
| Source name |
Sf9 cells transfected with AcMNPV bacmid carrying WT AAV2 insert
|
| Organism |
Spodoptera frugiperda |
| Characteristics |
treatment: 48h incubation
cell line: Sf9 insect cells
|
| Treatment protocol |
Two microgram of bacmid DNA were transfected into cells using Cellfectin II (Invitrogen) reagent according to the manufacturer instructions.
|
| Growth protocol |
1 million of Sf9 cells were plated on a 6 well plate with 2 mL of Sf900-III medium (Gibco #12658-027 1000 mL) and incubated at 27°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using RNeasy Mini Kit and enriched for Poly(A)-RNA with MicroPoly(A) Purist Kit.
The RNA was base-hydrolyzed, dephosphorylated with PNK and purified using RNA Clean & Concentrator kit (Zymo). Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers containing Illumina adapter sequences. Excess oligo was removed by Exonuclease I and cDNA fragments of were purified using ChIP DNA Clean & Concentrator kit. The recovered cDNA was RNaseH treated and circularized (CircLigase) and amplified for 10 cycles. The final product was ran on 10% TBE gel, gel purified (180-350 bp) and cleaned-up using ChIP DNA clean & Concentrator Kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
replicate 2
genome: Bacmid_dCC_AAV2wt
|
| Data processing |
Libraries were sequenced for 50 cycles on an Illumina HiSeq 2000 according to the manufacturer’s instructions.
RNA-Seq results were trimmed to remove A-stretches originating from the library preparation using by cutadapt and filtered for reads mapping to Sf21 genome
Alignment was performed using bowtie to the custom genomes.
Homer-software (http://homer.ucsd.edu/homer/) was used to prepare bedGraphs and quantify the reads for all annotations in reads per kb per million reads (RPKM).
Genome_build: custom fasta for genomes in supplementary files
|
| |
|
| Submission date |
Mar 21, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Minna U Kaikkonen |
| E-mail(s) |
minna.kaikkonen@uef.fi
|
| Organization name |
University of Eastern Finland
|
| Department |
A.I. Virtanen Institute, Department of Biotechnology and Molecular Medicine
|
| Street address |
P.O. Box 1627
|
| City |
Kuopio |
| ZIP/Postal code |
70211 |
| Country |
Finland |
| |
|
| Platform ID |
GPL17152 |
| Series (1) |
| GSE96855 |
Instability of WT AAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system |
|
| Relations |
| BioSample |
SAMN06624029 |
| SRA |
SRX2659259 |
