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Sample GSM2645391

Query DataSets for GSM2645391

Status

Public on Jan 05, 2018

Title

293_not_enough_24h_R1.fastq.gz

Sample type

SRA

Source name

HEK293 cells

Organisms

Caenorhabditis elegans; Drosophila melanogaster; Homo sapiens

Characteristics

cell line: HEK293 cells
treatment: 4SU
length of treatment: 24 hr
4su selected: TRUE
supplementary file: HEK293.notEnoughSpikeins.4SU.exon.csv
supplementary file: HEK293.notEnoughSpikeins.4SU.intron.csv
supplementary file: HEK293.notEnoughSpikeins.4SU.intron.preDRUID.csv
supplementary file: HEK.4SU.HL.DRUID.notEnoughSpikeIns.csv

Treatment protocol

For transcription inhibition experiments HEK293 were treated with either 5 µg/mL actinomycin D or 50 µg α-amanitin for 0, 1, 2, 4, 8, 12, and 24 hours. For metabolic labeling experiments cells were treated with 100 µM 4SU and harvested after 1, 2, 4, 8, 12, and 24 hr. HEK293 and S2 cells treated with 100µM 4SU for 24 h were used for the generation of labeled spike-ins. For 293_not_enough samples, HEK293 cells, were transfected for 2 hours with 1 µg/mL pUC19 using Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM (Thermo Fisher Scientific), 3 hours prior to the addition of 4SU. 1 hour prior to the addition of 4SU, media was changed and DMSO was added 1:2500.

Growth protocol

Human HEK293 epithelial cells (ATCC CRL1573) were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin solution. Murine NIH3T3 fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Donor Calf Serum (DCS) and 1% penicillin-streptomycin solution. Both mammalian cell lines were cultured at 37°C in a humidified incubator with 5% CO2. Drosophila melanogaster Schneider 2 (S2) cells were cultured in ExpressFive SFM media (Thermo Fisher Scientific) supplemented with 10% FBS (heat-inactivated) and 20mM L-Glutamine, at 28°C. Saccharomyces cerevisiae USY006 was grown in liquid YPD (20 g/L bacto peptone, 10g/L yeast extract, 950 mL water) at 30°C on a shaker at 200 rpm to mid-exponential phase.

Extracted molecule

polyA RNA

Extraction protocol

Following PBS washes, cell pellets were lysed in either TRI Reagent (Sigma-Aldrich) or Isol-RNA Lysis Reagent Manual (5Prime), according to manufacturer’s instructions. Briefly, 100 µL of 1-bromo-3-chloropropane was added to each sample, and samples were vortexed for 15 seconds and incubated at room temperature for 2 minutes. Sample were centrifuged in a prechilled centrifuge at 4°C at 15500 x g for 15 min. Aqueous phase was transferred to a new tube and 500µL isopropanol and 2µL glycoblue were added. Samples were vortexed for 10 s and then incubated for 10 min at room temperature. Samples were then centrifuged at 4°C at 15500 x g for 10 min. Supernatant was discarded and pellet was washed with 1 mL of 75% ethanol and centrifuged at 4°C, 9000 x g for 5 min. Ethanol was removed and pellet was resuspended in 20 µL Milli-Q water. For Saccharomyces cerevisiae 10 ml of the liquid culture was pelleted for 3 min at 1500 x g at 4°C. Supernatant was discarded and pellet was resuspended in 1 ml of ice-cold milli-Q water and pelleted again. The cell pellet was resuspended in 400 µL TES solution (10 mM Tris-Cl pH 7.5, 10mM EDTA, 0.5% SDS). 400 µL Phenol:Chloroform 5:1 was added, and the sample was vortexed vigorously for 10 s. After incubating at 65°C for 60 min with periodic vortexing, the sample was then centrifuged for 5 min at 21000 x g at 4°C. The aqueous phase was transferred to a new tube and reextracted with acid phenol and then again with chloroform. The aqueous phase was transferred, and RNA was precipitated with 40 µL of 3M sodium acetate, pH5.3 and 1 mL of ice-cold 100% ethanol. RNA was pelleted at 21000 x g for 5 min at 4°C. The RNA pellet was washed with ice-cold 70% ethanol and resuspended in 50 µL of milli-Q water. For Caenorhabditis elegans, sample volumes were brought up to 100 µL with UltraPure water and resuspended in 4X volume of TRI-Reagent. Samples were vortexed for 10 minutes at room temperature. 10% volume of 1-bromo-3-chloropropane was added and samples vortexed for an additional 10 minutes at room temperature. Samples were then centrifuge for 8 minutes at 10,000 x g at 4 degrees and top aqueous layer was transferred to a new tube. RNA was precipitated with 20 µg glycogen and 4X volume of 100% ethanol for at least 1 hour at -20°C. For transcription inhibition samples, 4 µg sample RNA was combined with 10 % Saccharomyces cerevisiae RNA. For metabolically labeled samples a 1 mg/mL solution of HPDP-biotin (Thermo Fisher Scientific) in dimethylformamide was incubated at 37°C for 30 minutes. 40-100 µg of RNA was combined with 20% w/w unlabeled yeast RNA (C. elegans N2 RNA for 293_not_enough samples gifted by the Claycomb lab), 20% w/w 4SU-labeled fly RNA and 120 µL of 2.5x citrate buffer (25 mM citrate pH 4.5, 2.5 mM EDTA) in a total volume of 240 µL. 60 µL of HPDP-biotin solution was added, and the solution was incubated at 37°C for 2 hr, covered and shaking at 200 rpm. Equal volume acid phenol was added, and samples were vortexed and separated at 21000 x g for 5 min at 4°C. Aqueous layer was re-extracted with acid phenol and again with chloroform. RNA was precipitated with 18 µL of 5M NaCl, 750 µL of 100% ethanol, and 2 µL glycoblue for a minimum of 60 min at –20°C. RNA was pelleted for 30 min at 21000 x g at 4°C and supernatural discarded. RNA pellet was resuspended in 200 µL 1x wash buffer (10 mM Tris-Cl pH 7.4, 50 mM NaCl, 1 mM EDTA). 50 µL MACS microbeads (Miltenyi Biotec) were incubated with 48 µL of 1x wash buffer and 2 µL yeast tRNA for 20 min at room temperature with rotation. The microcolumns (Miltenyi Biotec) were washed with 100 µL nucleic acid equilibration buffer (Miltenyi Biotec) and then five times with 100 µL 1x wash buffer. Beads were applied to the column in 100 µL aliquots and washed five times with 100 µL 1x wash buffer. Columns were demagnetized, and beads eluted with two 100 µL washes with 1x wash buffer. 200 µL of bead solution was combined with RNA sample and rotated at room temperature for 20 min. Sample was then applied to the remagnetized column in 100 µL aliquots. Columns were washed three times with 400 µL of wash 1 buffer (10mM Tris-Cl pH 7.4, 6M urea, 10mM EDTA) prewarmed to 65°C and then three times with 400 µL wash 2 buffer (10 mM Tris-Cl pH 7.4, 1M NaCl, 10mM EDTA). Selected RNA was eluted with five washes of 1x wash buffer with 0.1M dithiothreitol. RNA was precipitated for at least 1 hr at –20°C with 30 µL 5M NaCl, 1 mL ethanol, and 2 µL glycoblue and then pelleted for 30 min at 21000 x g and 4°C.
Sequencing libraries were prepared using the TruSeq Stranded mRNA Sample preparation kit (Illumina) per the manual (Rev. E).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Basecalling was performed using Illumina Real-Time Analysis v1.18.54 or later, and bcl2fastq2 v2.17 was used to convert to fastq format (by The Center for Applied Genetics (SickKids)).
Library quality was assessed using FastQC v0.11.5.
Reads were trimmed and clipped for Illumina adaptors using Trimmomatic v0.36 with the following options LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36.
Reads were aligned to merged reference genomes: hg38 (human) + sacCer3 (yeast), hg38 + dm6 (fly) + sacCer3, hg38 + dm6 + ce10 (worm), and mm10 (mouse) + dm6 + sacCer3 obtained using the UCSC Table Browser and kentUtils v302 on 08 Aug 2015. Reads were mapped using STAR version 2.5.2a_modified. STAR was used with default settings, aside from: outFilterMultimapNmax 10, outFilterMismatchNoverLmax 0.05, outFilterScoreMinOverLread 0.75, outFilterMatchNminOverLread 0.85, alignIntronMax 1, and outFilterIntronMotifs RemoveNoncanonical.
Mapped reads were quantified using two different methods. Firstly, an in-house R script using the GenomicFeatures and rtracklayer packages from Bioconductor and the plyr package was used to select the longest transcript for every gene and create gtf files containing exon and intron annotations. Any introns that overlapped with an exon in any other isoform were not included in the gtf. The htseq-count script from the HTSeq 0.6.1p2 was used to count reads mapping to introns and exons. In addition to HTSeq, an in-house intersection method based around the tools provided by BEDTools suite v2.26.0 was used. Briefly, rather than counting the number of reads mapping to a feature, coverage was determined at the nucleotide level and subsequently averaged at the feature level.
Genome_build: hg38, dm6, sacCer3, ce10
Supplementary_files_format_and_content: Comma separated values files containing: gene read counts and coverage determined for reads mapping to exonic and intronic regions for each gene in each sample, half-lives determined using different methods.

Submission date

May 31, 2017

Last update date

May 15, 2019

Contact name

Olivia Rissland

E-mail(s)

olivia.rissland@gmail.com

Organization name

The Hospital for Sick Children

Street address

686 Bay Street