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Sample GSM4104557

Query DataSets for GSM4104557

Status

Public on Mar 01, 2020

Title

CHO_0_hr_metabolic_labeling

Sample type

SRA

Source name

Chinese hamster ovary

Organism

Cricetulus griseus

Characteristics

cell line: CHO-K1
cell type: Chinese hamster ovary
atcc number: CCL-61
passages: 9
treatment: metabolic labeling
time: 0 hr

Treatment protocol

Cells were seeded into eleven 60mm dishes and pulse-labelled with 0.2 mM of 5-ethynyluridine (5-EU) at 40% confluency, keeping one plate not labelled for a control without 5-EU. 5-EU was chased after 24 hr of incorporation by adding 5 mM of uridine.

Growth protocol

Chinese hamster ovary cells (CHO-K1, ATCC CCL-61) were maintained in DMEM, high glucose, pyruvate, glutamine (Thermo Fisher Scientific 11995) supplemented with 10% Fetal Bovine Serum (FBS; Gibco 1992275), 1% Penicillin/Streptomycin, and 20 µg/mL L-proline (VWR AAA10199-14) and grown at 37°C with 5% CO2 supplementation.

Extracted molecule

total RNA

Extraction protocol

Cells were collected by replacing the media by 1 mL Trizol at the following time-points: 0 min, 30 min, 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr and 12 hr of chase. RNA was extracted following the Trizol extraction protocol, then re-extracted with phenol-chloroform as in. RNA was precipitated with sodium acetate and 100 % ethanol at -20°C, then spun down at 16,000 g for 20 min at 4°C, washed twice with 70 % EtOH and resuspended in water. DNAse I treatment was performed on duplicate RNA samples (2 x 6 µg) after addition of 5 ng of in vitro-labelled spike-in to each sample. The spike-in mix consisted of partial RNA sequences of B.subtilis LYSa and firefly luciferase transcripts, of which the gene sequences were cloned into pBluescript SK+ plasmid (pJC879 and pJC880, respectively) and transcribed using T7 RNA polymerase and 2 mM of 5-EU.
Samples were depleted of ribosomal RNAs using Illumina Ribo-Zero Gold rRNA removal kit (MRZG12324), then purified using the RNA Clean up and concentrator Kit (R1015, the epigenetics company). The duplicates for each sample were then combined before RNA fragmentation by alkaline treatment at 95°C for 25 min (NaCO3 pH9.2 50 mM final, EDTA 1 mM final). The reaction was immediately stopped on ice by addition of 0.3 M NaOAc pH5.2, 2µL glycoblue and 500µL water. RNA was precipitated with isopropanol, spun at 16,000 g for 30 min at 4°C, washed with 70 % EtOH and resuspended in 15.75 µL of water. Biotinylation of the 5-EU labelled RNA was performed following the Click-iT nascent RNA capture kit protocol and precipitated at -80°C overnight. The fragmented and biotinylated RNA was purified on NuPAGE denaturing polyacrylamide gels (TBE-urea 15 % polyacrylamide, Thermo Fisher Scientific EC62152BOX), excising RNA between 20 and 70 nucleotides. After extraction from the gel with 500 µL of RNA gel extraction buffer (300 mM NaOAc pH5.2, 1 mM EDTA, 0.25% (w/v) SDS) on a rotator at room temperature overnight, samples were precipitated with isopropanol and resuspended in 10 µL of 10 mM Tris-HCL pH8.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

CHO_FPKM.txt
CHO_Processed_Half_Lives.txt

Data processing

Supplementary_files_format_and_content: CHO_FPKM.txt - Tab-delimited text, FPKM calculated from CHO metabolic labeling
Supplementary_files_format_and_content: CHO_Processed_Half_Lives.txt - Tab-delimited text, CHO half-lives
Processing: Adapter sequence was clipped from raw reads (CTGTAGGCACCATCAAT) with fastx_clipper ver 0.0.13, and reads smaller than 18 nucleotides were discarded.
Mapping: The reads were then aligned to the Cricetulus griseus genome (GCF_000223135.1_CriGri_1.0) using hisat2 ver 2.1.0.
Downstream analyses: Sam files were converted into bam format and indexed using samtools v.1.7-2. Transcript FPKM values were calculated with Stringtie v.1.3.5 and Ballgown ver 2.16.0 (Pertea et al., Nat Protocol 2016) using default parameters and a gtf file of the CriGri_1 genome downloaded from RefSeq as annotation file. This file was used to create a merged transcripts annotation used in Stringtie to re-estimate the transcripts abundances that were given as input to Ballgown. The raw FPKM numbers at each time-point were normalized to the relative number of reads aligning to the spike-ins (average of reads aligning to Luc and LYSa normalized to the number of total reads) to adjust for the amplification resulting from a smaller pool of transcripts at the later time-points. Transcripts with less than 1 FPKM at time-point 0 and without a corresponding protein-coding sequence were filtered out. Half-lives were calculated as in Presnyak et al. 2015. Briefly, spike-in-normalized FPKM values for each timepoint were further normalized to the 0 hr value. Half-lives were estimated by fitting a least absolute deviations regression model. The decay equation model took into account the estimated doubling time of the cells (15 hr) to correct for the dilution due to cell growth. Resultant half-lives were filtered to exclude genes which had an estimated half-life longer than 18 hr and for which the average absolute residual was more than 20.
Genome_build: GCF_000223135.1, CriGri_1.0

Submission date

Oct 02, 2019

Last update date

Mar 01, 2020

Contact name

Jeff Coller

E-mail(s)

jmc71@case.edu

Phone

216-368-0299

Organization name

Case Western Reserve University