|
| Status |
Public on Apr 26, 2016 |
| Title |
CG3800 CLIPseq replicate input 2 |
| Sample type |
SRA |
| |
|
| Source name |
S2 cells expressing inducible CG3800
|
| Organism |
Drosophila melanogaster |
| Characteristics |
uv crosslinking: 254nm 400mJ/sqcm Spectrolinker
immunoprecipitated protein: FLAG/HA-tagged CG3800
adapter sequences: 3′ adapter (NNNN-TGGAATTCTCGGGTGCCAAGG) and 5′ adapter (GTTCAGAGTTCTACAGTCCGACGATC-NNNN)
|
| Treatment protocol |
For UV crosslinking, ~700*10e6 (at harvest) lowly passaged cells were cultured in ExpressFive SFM containing 100 microMolar Hygromycin B Gold solution and 300µM CuSO4 (last 18h before harvest) and collected by centrifugation (500×g for 5 min). Cells were washed once with ice-cold PBS and irradiated on ice at UV 254 nm (400mJ/sqcm) in a Spectrolinker XL-1000. Cross-linked cells were collected by centrifugation at 500×g for 5 min and immediately lysed in NP40 and sodium deoxycholeate (SDC) containing lysis buffer (1ml lysis buffer per 150*10e6 cells) (including RNAse inhibitors), incubated on ice for 10 min, and stored at -80°C.
|
| Growth protocol |
Drosophila S2 cells have been stably transfected with FMO-plasmides retrieved from The Drosophila Genomics Resource Center (DGRC) (PEP: FMO09119 and CG3800: FMO05319). After transfection, cells have been selected with Hygromycin B Gold solution (Invivogen) for > 5 passages and maintained at a concentration 100 micromolar Hygromycin in ExpressFive SFM medium containing 10% heat-inactivated fetal bovine serum (FBS) and supplemented with L-glutamine and Penicillin-Streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
CLIPseq was performed in biological replicates and was essentially described in (Hafner et al. 2010). Buffers and RNA digest were adapted from (Huppertz et al. 2014). The cell lysate was thawed on ice and RNAseI (333U/ml lysate; 1:333 (v/v)) and TurboDNAse (4U/ml lysate) treated for 3.5min at 37°C and 1100rmp and immediately replaced on ice for >3min. Lysates were cleared by centrifugation at 20,000g at 4°C. Immunoprecipitation (IP) was carried out with monoclonal FlagM2 (SIGMA) coated magnetic proteinG dynabeads (Life) on spin-cleared cell extracts for 2 h at 4ºC. Before IP, 50µl of the cleared lysate was taken, proteinase K treated and used for RNA extraction (Zymo DirectZol RNA-Mini Prep Kit). 2.5µg of this fragmented input total RNA was Ribozero treated. 100ng of rRNA-depleted, radioactively end-labeled input RNA was used to construct CLIP input libraries and resumed to CLIP cloning procedure at the step of 3’adapter ligation and was therefore subjected to the same RNA fragment size selection and adapter ligation procedures (see below). After IP, CLIP samples were stringently washed 3 times with high salt buffer (1M NaCl). Samples were radioactively end-labeled with γ-32P-ATP (25min at 37°C and 1100rpm) and subsequent addition of 1µl high molar ATP for efficient 5’end phosphorylation. The crosslinked protein-RNA complexes were resolved on a 4-12% NuPAGE gel (Invitrogen). The SDS-PAGE gel was transferred to a nitrocellulose membrane (BioRad) and the protein-RNA complex migrating at an expected molecular weight was excised. RNA was isolated by Proteinase K (Roche) treatment and phenol-chloroform extraction, ligated to 3′ adapter (NNNN-TGGAATTCTCGGGTGCCAAGG) and 5′ adapter (GTTCAGAGTTCTACAGTCCGACGATC-NNNN), reverse transcribed, PCR-amplified (PCR cycles for libraries: PEP_CLIP_R1 und R2 18 x cycles; PEP_INPUT_R1 und R2 10 x cycles; CG3800_CLIP_R1 und R2 14 x cycles, CG3800_INPUT_R1 und R2 10 x cycles), and gel-excised. The amplified PEP cDNA was sequenced on HighSeq2000 (Illumina) with a 1x51 nt cycle. CG3800 cDNA was sequenced on NextSeq500 (Illumina) with 75 cycles single-end high output.
Each CLIP was conducted in a biological replicate including a sized-matched input RNA library.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
RNAseI-treated, ProteinaseK-treated, rRNA-depleted, size-selected (19-50nt) total RNA
|
| Data processing |
none provided by the submitter
|
| |
|
| Submission date |
Feb 24, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hans-Hermann Wessels |
| E-mail(s) |
[email protected]
|
| Organization name |
New York Genome Center
|
| Street address |
101 Avenue of the Americas
|
| City |
New York |
| ZIP/Postal code |
10013 |
| Country |
USA |
| |
|
| Platform ID |
GPL19132 |
| Series (1) |
| GSE78237 |
The mRNA-bound proteome of the early fly embryo |
|
| Relations |
| BioSample |
SAMN04511097 |
| SRA |
SRX1598176 |
