 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 03, 2023 |
| Title |
Ded1-500nM_b |
| Sample type |
SRA |
| |
|
| Source name |
yeast cell
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell line: BY4741
cell type: yeast strain
genotype: MATa his3{delta}1 leu2{delta}0 met15{delta}0 ura3{delta}0
treatment: Ded1-500nM
|
| Treatment protocol |
No additional treatment for the cell.
|
| Growth protocol |
WT yeast strain was cultured in Yeast Extract–Peptone–Dextrose (YPD) medium to OD600 of ~1. Harvested cells were washed with cold water and stored in -80°C before use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The frozen cell pellet from a 500 ml culture was thawed on ice and mechanically disrupted by vortexing with glass beads three times for 2 min each at maximum speed in a cold room (4°C) in 2 ml of buffer RL (provided in the kit), 3 ml phenol:chloroform:isoamyl alcohol 25:24:1 (pH 5.2) and 3 ml ice cold glass beads. Following 5 min centrifugation at 14000 rpm in a Sorvall LYNX 6000 Superspeed centrifuge using a Fiberlite F14-14 x 50cy rotor, the supernatant was transferred to a fresh tube and total RNA was prepared using a GenElute total RNA purification Maxi kit (Sigma-Aldrich; RNB200) following the manufacturer’s protocol. 400 μg total RNA in 250 μl water was applied to a GeneElute mRNA miniprep kit (Sigma-Aldrich; MRN70-1KT), following the manufacturer’s protocol. The purified total mRNA was treated by 5’-Phosphate-Dependent Exonuclease (Lucigen, TER51020) to degrade RNAs with 5’ monophosphates, such as 18S and 25S ribosomal RNAs, at 0.3 μg/µl mRNA in 1X buffer A (provided in the kit), 1U/µl RiboGuard RNase Inhibitor (Lucigen; RG90925), 0.1 U/µl 5’-Phosphate-Dependent Exonuclease. Following a 1 h incubation at 30°C, the total mRNA was extracted using phenol:chloroform:isoamyl alcohol 25:24:1 (pH 5.2), ethanol precipitated, resuspended in 10 mM Tris (pH 8.0) and stored in -80 °C.
RecSeq sequencing library construction was conducted according to a previously described protocol (McGlincy and Ingolia 2017), with modifications, using identical barcoded linkers (NI-810 to NI-815), RT primer (NI-802) and PCR primers (NI-798, NI-799, NI-822 to NI-824). The RNA fragments purified from 48S PIC RPFs were dephosphorylated using T4 Polynucleotide Kinase (PNK) (NEB; M0201L) and ligated to pre-adenylated linkers (NI-810 to NI-815) containing 5 nt sample barcodes unique for each sample using truncated T4 RNA ligase 2 (K227Q) (NEB; M0351L). Ligated fragments were separated from free linkers on a 15% polyacrylamide TBE-Urea gel and then pooled and purified for reverse transcription using RT primer NI-802 and ProtoScript II Reverse Transcriptase (NEB; M0368S). The ~105 nt cDNAs were separated from free RT primers on a 15% TBE-Urea gel and circularized using CircLigaseII ssDNA Ligase (Biosearch Technologies; CL9021K). PCR was carried out using forward primer NI-798, and reverse primers (NI-799, NI-822 to 824) as already described (McGlincy and Ingolia 2017) .
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
48S Preinitiaiton complex protected mRNA fragments
|
| Data processing |
Remove sequencing adaptors sequence (AGATCGGAAGAGCAC) in the fastq files using cutadapt/4.4.
Demultiplex fastq sequences using Linux "grep" function to subset reads containing sample specific barcode at the 3' ends.
Remove non-coding RNA reads using STAR/2.7.10b
Map non-coding RNA removed reads to yeast genome using STAR/2.7.10b, and count 25-34 nt reads mapped to each transcript using "fp-count" function in RiboSeq (https://github.com/ingolia-lab/RiboSeq)
Map non-coding RNA removed reads to spike-in sequences using STAR/2.7.10b, and count 25-34 nt reads mapped to each transcript using "fp-count" function in RiboSeq (https://github.com/ingolia-lab/RiboSeq).
Wiggle files were produced from the alignment files using "wiggle-track" function in RiboSeq (https://github.com/ingolia-lab/RiboSeq), generating two wiggle files, each for genes on the Watson or Crick strand. Wiggle files were normalized by multiplying the size factor "q" (calculate using spike-in RPFs) in the wiggle-track function (-q).
Differential expression analysis of changes in RPF, recruitment efficiency (RE), or relative ribosome occupancy (RRO) values was performed using DESeq2 (Love et al., 2014) using custermerized Size-factors calculated from spike-in RPFs.
Assembly: R64-1-1 S288C Sac cer3 Genome Assembly
Supplementary files format and content: Wiggle files
Library strategy: Rec-Seq
|
| |
|
| Submission date |
Sep 26, 2023 |
| Last update date |
Oct 03, 2023 |
| Contact name |
Jon R. Lorsch |
| E-mail(s) |
[email protected]
|
| Phone |
301-594-2172
|
| Organization name |
National Institutes of Health
|
| Department |
Eunice Kennedy Shriver National Institute of Child Health and Human Development,
|
| Lab |
Section on the Mechanism and Regulation of Protein Synthesis
|
| Street address |
BG 49 RM 2C08, 49 Convent Dr.
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL27812 |
| Series (1) |
| GSE244093 |
Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system |
|
| Relations |
| BioSample |
SAMN37547833 |
| SRA |
SRX21896509 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7806550_Ded1_500nM_b_fwd.wig.gz |
161.3 Kb |
(ftp)(http) |
WIG |
| GSM7806550_Ded1_500nM_b_rev.wig.gz |
158.8 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
