|
| Status |
Public on Feb 20, 2021 |
| Title |
MmGtsf1-Strep in Sf9 - rip1_2 |
| Sample type |
SRA |
| |
|
| Source name |
Sf9
|
| Organism |
Spodoptera frugiperda |
| Characteristics |
cell line: Sf9
pull-down: Strep-Tactin beads pull-down
|
| Treatment protocol |
Asterix/Gtsf1 protein from mouse with a C-terminal TEV-Strep tag was expressed recombinantly using the MultiBac expression system. Briefly, the cDNA corresponding to the mouse Gtsf1 protein was cloned into a vector (pFL) that allows for bacmid incorporation and subsequent baculovirus generation upon transfection. The baculovirus was amplified after transfection through additional rounds of infection and medium harvesting. For large-scale expression, cells were harvested 72 hours post-infection.
|
| Growth protocol |
Sf9 cells were maintained in HyClone CCM3 medium and grown in suspension at 27°C with shaking. Cultures were monitored for Mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). Mycoplasma contamination was not detected.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed by sonication in 50 mM Tris, pH 8.0, 100 mM KCl, 1 mM DTT, then purified by affinity chromatography using Strep-Tactin resin (IBA). The purified Gtsf1 protein and co-purifying nucleic acids were eluted in lysis buffer supplemented with 5 mM d-desthiobiotin. The eluted material was further purified by ion exchange chromatography (MonoQ column) and nucleic acid containing fractions were taken for library construction.
Small RNA libraries were prepared using the SMARTer smRNA-Seq Kit for Illumina sequencing (Takara). In addition to the co-purifying RNAs, total Sf9 RNAs were similarly processed as input controls. Size-selection was performed using Blue Pippin 2% agarose gel cassettes (Sage Science). All libraries were assessed by fluorometric quantification (Qubit 3.0) and by Bioanalyzer chip-based capillary electrophoresis.
Libraries were pooled in equimolar ratios according to their quantification by Bioanalyzer. Single-end reads with two 8-basepair barcodes were generated on an Illumina NextSeq resulting in approximately 10 million reads per library.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Basecalling was performed with bcl2fastq2 v2.19 (Illumina).
Reads were trimmed to remove the first 3 nucleotides (contributed by the kit chemistry) and to remove low quality ends with trimmomatic. -threads 1 -phred33 fastq_in.fastqsanger fastq_out.fastqsanger HEADCROP:3 SLIDINGWINDOW:4:20
polyA tails (also contributed from the kit chemistry) were removed using a custom script.
Size selection was performed to retain reads between 30 and 85 nucleotides in length using Galaxy.
Reads with poor quality were filtered out using Galaxy. 95% of bases were required to have a Q-value over 24 to be retained.
To assess the most abundant reads, identical reads were counted and collapsed using Galaxy.
Genome_build: no mapping performed due to low quality genome assembly
Supplementary_files_format_and_content: *_final_collapsed.fasta; fasta-formatted reads, collapsed based on counts of sequencing reads that are identical after processing. The reads are sorted from most common to least common (from top to bottom) with identifier rows indicating the read number (1, 2, 3, etc.) and number of instances of that read (e.g. >6-36055 indicates the sixth most abundant sequence, being represented 36055 times in the library).
|
| |
|
| Submission date |
May 24, 2020 |
| Last update date |
Feb 22, 2021 |
| Contact name |
Jonathan James Ipsaro |
| E-mail(s) |
jipsaro@cshl.edu
|
| Organization name |
Cold Spring Harbor Laboratory
|
| Lab |
Leemor Joshua-Tor
|
| Street address |
1 Bungtown Road
|
| City |
Cold Spring Harbor |
| State/province |
NY |
| ZIP/Postal code |
11724 |
| Country |
USA |
| |
|
| Platform ID |
GPL27985 |
| Series (2) |
| GSE151110 |
RIP-seq of mouse Asterix/Gtsf1 expressed in Sf9 cells |
| GSE151112 |
Asterix/Gtsf1 links tRNAs and piRNA silencing of retrotransposons |
|
| Relations |
| BioSample |
SAMN15006193 |
| SRA |
SRX8387826 |
