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Sample GSM8410985

Query DataSets for GSM8410985

Status

Public on Sep 11, 2024

Title

DRS_Pab1_AID_25°C_1h_repA

Sample type

SRA

Source name

W303

Organism

Saccharomyces cerevisiae

Characteristics

genotype: Pab1-AID
strain: W303
treatment: 25degC + auxin for 1h

Treatment protocol

To deplete Mex67 using the Anchor Away tag (Haruki et al, 2008), rapamycin (Cayman Chemicals cat. no. 13346) was added to a final concentration of 1 μg/ml. The heat-stress chase experiment was performed by pre-culturing cells at 25 °C and adding an equal volume of media pre-heated to 51 °C, resulting in a final temperature of 37 °C. Treatment with thiolutin (Sigma; T3450) was performed by adding the compound to a final concentration of 4 ug/ml. Samples for all chase experiments were collected by mixing the cell culture with an equal volume of ice-cold ethanol, which inactivates cellular metabolism. To deplete Xrn1 and Dcp2 using AID tags (Nishimura et al, 2009; Morawska and Ulrich, 2013), 1-Naphthaleneacetic acid (1-NAA; N0640-25G, Merck) was added to a final concentration of 1 mM. Pab1 depletion using the AID system involved adding auxin sodium salt (I5148-2G; Merck) to a final concentration of 1-3 mM

Growth protocol

Yeast cultures were prepared in YPDA media and grown in 25 °C unless stated othwerwise

Extracted molecule

polyA RNA

Extraction protocol

RNA extraction was performed using the hot acid phenol method. Cell pellets were resuspended in 400 μl phenol solution saturated with 0.1 M citrate at pH 4.3 (Sigma; P4682), followed by the addition of 400μl of TES buffer (10 mM Tris pH 7.5, 5 mM EDTA, 1 % SDS). Samples were vortexed for 45 min at 65 °C, then centrifuged at 4 °C for 10 min. The supernatant was transferred to a fresh tube, and 400 μl phenol solution saturated with 0.1 M citrate at pH 4.3 was added. The samples were again vortexed for 20 min at 65 °C and then centrifuged at 4 °C for 10 min. The supernatant was transferred to a fresh tube and 400 μl of chloroform was added (C2432; Sigma). The samples were briefly vortexed at room temperature to remove phenol, and centrifuged at 4 °C for 10 min. The supernatant was transferred to a fresh tube; 45 μl of 2 M LiCl was added and 1 ml of 95 % ethanol. Samples were precipitated at – 80 °C for at least 30 min, then centrifuged at 4 °C for 25 min, washed with 400 μl of 80 % ethanol, and dried at 37 °C after removing the supernatant. Pellets were resuspended in nuclease-free water, and RNA concentration was measured using a Nanodrop apparatus.
The pA+ fraction was prepared using magnetic beads coupled to oligo-dT from LifeTechnologies (61005). 35 μg of total RNA was resuspended in 50 μl of nuclease-free water. The RNA was mixed with 50 μl of binding buffer (20 mM Tris-HCl, ph 7.5, 1 M LiCl, 2 mM EDTA) and denatured for 2 min at 65 °C before cooling on ice. 100 μl of slurry beads per 35 μg of total RNA was pre-washed 3 times in 1 ml of binding buffer and resuspended in 50 μl of binding buffer per sample. The beads were added to the denatured RNA and incubated at room temperature with occasional shaking for 20 min. The supernatant was removed, and beads were washed twice with 100 μl of wash buffer (10 mM Tris-HCl pH 7.5, 150 mM LiCl, 1 mM EDTA) and after removing any remnants of wash buffer, resuspended in 12 μl of nuclease-free water. The beads were then incubated for 2 min at 80 °C, and the supernatant removed from the beads was utilized for sequencing library preparation and/or qPCR analyses as the pA+ fraction. Sequencing libraries were prepared using the Direct RNA Sequencing Kit (Oxford Nanopore Technologies, SQK- RNA002) according to the manufacturer’s protocol, using 50–200 ng oligo-dT(25)-enriched mRNA from Saccharomyces cerevisiae yeast mixed with cap-enriched or total RNA from other organisms (human, mouse, A. thaliana, or C. elegans)
RNA-seq (Nanopore Direct RNA Sequencing)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Description

Nanopore Direct RNA Sequencing of S. cerevisaie W303, Pab1-AID degron, grown at 25°C, treated with auxin for 1h, experiment A

Data processing

Sequencing was performed with the MinION device, followed by basecalling with Guppy (ONT), mapping to custom S. cerevisiae transcriptome described in Tudek et al (2021) using Minimap2 2.17 with options -k 14 -ax map-ont –secondary = no, and processing with samtools 1.9 to filter out supplementary alignments and reads mapping to the reverse strand (samtools view -b -F 2320). Lengths of poly(A) tails were estimated with Nanopolish-polya (v0.13.2).
Assembly: SacCer3
Supplementary files format and content: CSV files with the experiment-related information on nanopolish-polya derived poly(A) lengths (including mean, meadina values and quantiles of distribution) and counts attributed to each transcript

Submission date

Jul 22, 2024

Last update date

Sep 11, 2024

Contact name

Andrzej Dziembowski

E-mail(s)

adziembowski@iimcb.gov.pl

Organization name

International Institute Of Molecular And Cell Biology In Warsaw

Lab

Laboratory of RNA Biology