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Sample GSM8278625

Query DataSets for GSM8278625

Status

Public on Jul 10, 2024

Title

RNA_pellet_1

Sample type

SRA

Source name

HEK293

Organism

Homo sapiens

Characteristics

cell line: HEK293

Extracted molecule

total RNA

Extraction protocol

HEK cells were cultured in a 10 cm dish in preparation for the RocA treatment, and in a 6-well plate for the heat shock treatment. The cells were then lysed in 500 µl and 100 µl of lysis buffer. Human skin cells cultured in a well of 6-well plates were lysed in 100 μl of lysis buffer. The mouse forebrain was dissected on ice and disrupted using a loose fitting Dounce homogenizer in 3 mL ice-cold homogenizing buffer.The homogenate was treated with an equal volume of 2× lysis buffer and incubated for 10 min on ice. S2 cells, cultured for 3 days, were seeded in 10 cm dishes. To these cells, 10 μl of 100 mg/ml cycloheximide was added, followed by immediate centrifugation at 1,000 × g to collect the cell pellet. The pellet was then washed with PBS and treated with 400 μl of lysis buffer. E. coli MG1655 cells grown overnight at 37°C in LB media were diluted 1:100 in fresh LB media and grown at 37°C until the OD600 was 0.4-0.5. Cells were then collected by filtration and snap frozen in liquid nitrogen. The frozen cell pellet and lysis buffer were disrupted using a multi-beads shocker. For yeast, cells collected in ribosome profiling lysis buffer were pulverised using a Multi-Beads Shocker with a pre-cooled chamber and ball using liquid nitrogen. 0.2 g of frozen Arabidopsis wild-type (Col-0) seedlings were combined with 400 μl of Arabidopsis lysis buffer.This mixture was then subjected to grinding using a multi-bead shocker
For ribosome profiling, RNase I treatment, ribosome pelleting, size selection, preadenylated linker ligation, rRNA depeletion, reverse transcription, cDNA circularization, and PCR amplification..For RNA-Seq, TruSeq Stranded mRNA Library Prep Kit (Illumina) or SMARTerseq (Illumina). For Escherichia coli, MNase footprinting, ribosome pelleting, RNA extraction, preadenylated linker ligation, rRNA depeletion, reverse transcription, cDNA circularization, PCR amplification (for ribosome profiling)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq X Plus

Description

RNA-seq_and_rRNA_depletion_with_ribopools

Data processing

Basecalling with Illumina Casava 1.8 software
3' adapter trimming with Fastp
Random barcode triming with custom script
Alignment to rRNA and other non-coding RNA with STAR v2.7.0a
Alignment to genome with STAR v2.7.0a
Read quantitation using custom scripts
Assembly: hg38,mm10,SacCer-3,dm6,TAIR10,NC_000913.2
Supplementary files format and content: text files contain three columns: 1. transcript name; 2. CDS region (nt) used for read counting; 3. read counts
Supplementary files format and content: csv files contain mapped reads and depleted reads information as indicated in the colunms

Submission date

May 17, 2024

Last update date

Jul 10, 2024

Contact name

Shintaro Iwasaki

E-mail(s)

[email protected]

Organization name

RIKEN

Department

CPR