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Sample GSM4477455

Query DataSets for GSM4477455

Status

Public on Jun 25, 2020

Title

Total 40S Ribosome FPs Hela cells without crosslink [7_40S_Total_UT]

Sample type

SRA

Source name

HeLa

Organism

Homo sapiens

Characteristics

cell line: HeLa
rrna depletion: Custom rRNA Depletion
library preparation kit: Takara
index name: R8
index sequence: TAATGCGC

Treatment protocol

Cellular stress conditions were induced by treatment with Tunicamycin at 1 µg / ml or 250 ng / ml for 16 hours.

Growth protocol

HeLa cells were cultured in DMEM +10% fetal bovine serum +100 U/ml Penicillin/Streptomycin (Gibco 15140122).

Extracted molecule

total RNA

Extraction protocol

For cell harvest, growth medium was poured off and cells were quickly washed with ice-cold washing solution (1x PBS 10 mM MgCl2 800 µM Cycloheximide). Washing solution was immediately poured off and freshly prepared crosslinking solution (1x PBS, 10 mM MgCl2, 200 µM Cycloheximide, 0.025% PFA, 0.5 mM DSP) was added to the cells. Cells were incubated with crosslinking solution for 15 minutes at room temperature while slowly rocking. Crosslinking solution was then poured off and remaining crosslinker was inactivated for 5 minutes with ice-cold quenching solution (1x PBS, 10 mM MgCl2, 200 µM Cycloheximide, 300 mM Glycine). Quenching solution was poured off and 150 µl of lysis buffer (0,25 M HEPES pH 7.5, 50 mM MgCl2, 1 M KCl, 5% NP40, 1000 μM CHX) was added to each 15 cm dish, resulting in 750µL of lysate. Lysis was carried out at 4°C.
After RNA extraction from total and IP-purified fractions, RNA quality and integrity were determined on an Agilent Bioanalyzer using the total RNA Nano 6000 Chip. For size selection, RNA was run on 15% Urea-Polyacrylamide gels (Invitrogen) and fragments of size 20-60 nt (80S libraries) and 20-80 nt (40S libraries) were excised using the Agilent small RNA ladder as a reference. RNA was extracted from the gel pieces by smashing the gels into small pieces with gel smasher tubes and extracting the RNA in 0.5 ml of 10 mM Tris pH 7 at 70°C for 10 minutes. Gel pieces were removed and RNA was precipitated using Isopropanol. Footprints were then dephosphorylated using T4 PNK (NEB) for 2 hours at 37°C in PNK buffer without ATP. Footprints were then again precipitated and purified using isopropanol. For 40S footprints, contaminating 18S rRNA fragments were depleted as follows. Prevalent 18S rRNA fragments from the first round of 40S footprinting were used to design complementary Biotin-TEG-DNA oligonucleotides (sequences listed in Supplemental Table 4, ordered from Sigma-Aldrich). 100ng of RNA footprints were then hybridized to a mixture (proportional to occurrence of the fragment, listed in Suppl. Table 4) of these DNA oligos (in 40x molar excess) in (0.5M NaCl, 20mM Tris pH7.0, 1mM EDTA, 0.05% Tween20) by denaturing for 90 sec at 95C and then annealing by reducing the temperature by -0.1C/sec down to 37°C, then incubating 15min at 37°C. Hybridized species were pulled out using Streptavidin magnetic beads (NEB) by incubating at room temperature for 15 minutes, and the remaining RNA was purified by isopropanol precipitation. Footprints were then assayed using an Agilent Bioanalyzer small RNA chip and Qubit smRNA kit. 25 ng or less of footprint RNA was used as input for library preparation with SMARTer smRNA-SeqKit for Illumina from Takara / Clontech Laboratories according to the manufacturer’s instructions. Deep-sequencing libraries were sequenced on the Illumina Next-Seq 550 system.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

NextSeq 550

Description

40S_Total_Untreated(No Crosslink)
processed data file: Master_Data_Table.xlsx

Data processing

Library strategy: Ribo-seq
Adapter sequences and randomized nucleotides (Nextflex) or polyA stretches (Clonentech) were trimmed from raw reads using cutadapt.
Nuclear and mitochondrial Ribosomal RNA and tRNA reads were removed by alignment to human tRNA and rRNA sequences using bowtie2.
The remaining reads were separately aligned to the human transcriptome (Ensemble transcript assembly 94) and human genome using BBmap. Multiple mappings were allowed.
Genome_build: human transcriptome (Ensemble transcript assembly 94)
Supplementary_files_format_and_content: Master_Data_Table1.xlsx: Excel file includes values that are normalized to sequencing depth (divided by number of mappings and multiplied by a scaling factor).

Submission date

Apr 15, 2020

Last update date

Jun 25, 2020

Contact name

Jonathan Bohlen

Organization name

DKFZ

Department

B140

Lab

AG Teleman