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Status |
Public on Dec 02, 2023 |
Title |
MitoticArrest TIS Input RNA rep 1 |
Sample type |
SRA |
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Source name |
HeLa
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Organism |
Homo sapiens |
Characteristics |
cell stage: Mitotic Arrest treatment: Harringtonine-puromycin replicate: 1 library type: mRNA
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Treatment protocol |
For mitotic arrest synchronization, HeLa cells were grown to 40% confluency and treated with 2mM thymidine for 24 hours. Cells were released from thymidine arrest by washing twice with warm DMEM and cultured in 8 µM STLC for 16 hours. Mitotically arrested cells were shaken off the plate and harvested. For cycling mitotic cells, HeLa cells were grown to 20% confluency and treated for 2 mM thymidine for 16 hours. Cells were then washed twice with warm DMEM, incubated at 37°C for 8 hours then 2 mM thymidine was added for another 16 hours. After washing twice with warm DMEM and incubation at 37°C for 8 hours, the cycling mitotic cells were shaken off and harvested. For interphase cells, the mitotic cells were removed by physical disruption (mitotic shake off) and the remaining cells adhered to the plate were harvested. For elongating ribosome profiling, cells were treated with 100 μg/mL cycloheximide for 2 minutes then harvested. For initiation site sequencing, cells were treated with 2 μg/mL harringtonine for 2 minutes at 37°C, followed by 12.5 μM puromycin for 2 minutes at 37°C then 100 μg/mL cycloheximide and immediately collected.
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Growth protocol |
HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin.
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Extracted molecule |
total RNA |
Extraction protocol |
To collect interphase cells, media containing translation inhibitor was poured into a 50mL tube, cells were washed with PBS + 100 μg/mL cycloheximide and then trypsinized in the presence of 100 μg/mL cycloheximide for 3 minutes at 37°C. Cells were then collected using the cold media with inhibitor. For mitotic cells, after addition of cycloheximide, mitotic cells were harvested by shake off and incubated on ice. Cells were centrifuged at 500x g for 3 minutes, washed once with 20 mL PBS+cycloheximide, and the pellet was moved into a new 1.5 mL tube with 1 mL PBS+cycloheximide. Cells were lysed in 900 μL polysome lysis buffer (20 mM Tris pH 7.4, 100 mM KCl, 5 mM MgCl2, 1% (v/v) Triton X-100, 100 μg/mL cycloheximide, 500 U/mL RNaseIn Plus, 1x cOmplete protease inhibitor cocktail) and incubated on ice for 10 minutes. To ensure efficient lysis, cells were passed through a syringe 5 times. Cell debris and nucleus was depleted by spinning the lysate at 1300x g for 10 minutes. 30 μL of the cleared lysate was added to 400 μL TRI Reagent (Invitrogen, AM9738) for input RNA sequencing library. Three 300μL aliquots were generated with the rest of the cleared lysate, flash frozen in liquid nitrogen, and stored at -80°C.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
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Description |
CellCycle_TIS_input_counts.txt
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Data processing |
Samples were demultiplexed with bcl2fastq v2.20 to generate fastq files. Read trimming: Cutadapt (v3.7). cutadapt -m 15 -u 8 -e 0.1 --match-read-wildcards -a TCGTATGCCGTCTTCTGCTTG -O 1 Read mapping: STAR (v2.7.1a): STAR --runMode alignReads --outFilterMultimapNmax 1 --outReadsUnmapped Fastx --outFilterType BySJout --outSAMattributes All --outSAMtype BAM SortedByCoordinate The RiboTISH pipeline was applied to aligned reads from both translation initiation site sequencing and elongating ribosome profiling to identify high confidence translation initiation sites. Read counting: htseq-count (0.11.0): For elongating ribosome profiling and matched RNA-seq: htseq-count -f bam -t CDS. Assembly: hg38, Gencode release 25 annotations. Supplementary files format and content: Tab delimited files with chromosome position for the translation initiation site and counts at each site along with the associated input mRNA counts is provided for each TIS-seq dataset. Tab delimited file with gene ID and counts within the annotated open reading frames is provided for elongating ribosome profiling and input mRNA sequencing datasets. Library strategy: Ribosome profiling
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Submission date |
Apr 20, 2023 |
Last update date |
Dec 02, 2023 |
Contact name |
Jimmy Ly |
Organization name |
Whitehead Institute
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Department |
Biology
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Lab |
Iain Cheeseman
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Street address |
455 main street
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City |
Cambridge |
State/province |
Massachusetts |
ZIP/Postal code |
02142 |
Country |
USA |
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Platform ID |
GPL16791 |
Series (1) |
GSE230189 |
Nuclear release of eIF1 restricts start-codon selection during mitosis |
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Relations |
BioSample |
SAMN34268243 |
SRA |
SRX20027533 |
Supplementary file |
Size |
Download |
File type/resource |
GSM7191434_AGCGCT_STLCHarrInput_Rep1.bedgraph.gz |
23.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
Processed data are available on Series record |
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