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Sample GSM6914611

Query DataSets for GSM6914611

Status

Public on Jan 04, 2023

Title

NAI-KD1_1-F1R1_F3R3

Sample type

SRA

Source name

HeLa cells

Organism

Homo sapiens

Characteristics

cell line: HeLa
shRNA: shURB1_4
treatment: NAI samples modified by NAI in DMSO

Treatment protocol

Scramble and URB1-targetting shRNA expressing HeLa cells were were seeded and induced with 1 μg/ml doxycycline for 6 days.

Growth protocol

HeLa cells expressing scramble tet-on shRNA or URB1-targetting tet-on shRNA were maintained in DMEM, which was supplemented with 10% Fetal Bovine Serum (FBS).

Extracted molecule

total RNA

Extraction protocol

The Tet inducible stable URB1-KD HeLa cell lines were seeded and induced with 1μg/mL doxycycline on 6 cm dishes for 6 days. After washed with DPBS, cells were incubated with 270 μL of cell culture medium and 30 μL of 10x SHAPE Chemical in DMSO with the final concentration of NAI (EMD Millipore) at 100 mM for 10 minutes at 37 °C. After removed reaction medium, RNAs were isolated by 1 mL Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The same procedure was also performed in parallel for the untreated control samples, but with the addition of only DMSO. In the denaturing control (DC) reaction, RNAs were suspended in a denaturing buffer containing 50% formamide and were incubated at 95 °C before modification with SHAPE reagents. For DMS samples, after washed with DPBS, cells were treated with the addition of 15uL DMS and incubation at 37°C for 5min. Cell culture media with DMS was slowly decanted. Then cells were washed twice in 30% v/v BME (diluted in DPBS) and collected into a 15ml centrifuge tube using a scraper. Centrifuge cells at 1000 × g at 4°C for 3min and decant the BME solution. Wash the cells with 1ml DPBS twice and centrifuge the cells again. Isolated RNAs were treated with DNase I (Ambion, DNA-freeTM kit) to remove possible DNA contamination. About 50-100 ng of RNAs were obtained under each treatment and were then used for SHAPE-MaP reverse transcription by adding 1 mL (200 U/mL) of SuperScript II (Invitrogen), 6 mM Mn2+ and gene-specific primers for 3' ETS. Second-strand synthesis was performed with TAQ DNA polymerase. The resulting PCR products were further isolated. Primers for SHAPE-MaP reverse transcription and second-strand synthesis (1st round PCR and nested PCR reactions) were listed in Supplementary Table S1. SHAPE-MaP libraries were prepared from 1 ng of DNAs, and size-selected with AmpureXP beads (Agencourt) with a 1:1 (bead to sample) ratio to obtain library DNA products spanning 100-400 bp in length. Final libraries were quantified using Agilent Bioanalyzer 2100 and QuBit high-sensitivity dsDNA assay. Deep sequencing was performed by Illumina NextSeq 500 at Shanghai Institute Nutrition and Health, Big Data Center Omics Core, Shanghai, China. About 15-25 million mapped sequencing reads were obtained for each sample, with 88% of bases at or above Q30.
Isolated RNAs were treated with DNase I (Ambion, DNA-freeTM kit) to remove possible DNA contamination. About 50-100 ng of RNAs were obtained under each treatment and were then used for SHAPE-MaP reverse transcription by adding 1 mL (200 U/mL) of SuperScript II (Invitrogen), 6 mM Mn2+ and gene-specific primers for 3' ETS. Second-strand synthesis was performed with TAQ DNA polymerase. The resulting PCR products were further isolated. Primers for SHAPE-MaP reverse transcription and second-strand synthesis (1st round PCR and nested PCR reactions) were listed in Supplementary Table S1.
SHAPE-MaP libraries were prepared from 1 ng of DNAs, and size-selected with AmpureXP beads (Agencourt) with a 1:1 (bead to sample) ratio to obtain library DNA products spanning 100-400 bp in length. Final libraries were quantified using Agilent Bioanalyzer 2100 and QuBit high-sensitivity dsDNA assay. Deep sequencing was performed by Illumina NextSeq 500 at Shanghai Institute Nutrition and Health, Big Data Center Omics Core, Shanghai, China. About 15-25 million mapped sequencing reads were obtained for each sample, with 88% of bases at or above Q30.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

NAI-KD1_1-F1R1_F3R3_F4R4_profile.txt

Data processing

Basecalling using Illumina bcl2fastq v2.17.1.14 software.
High-throughput sequencing reads were separated according to 6-nt experimental barcodes.
Raw read was evaluated quality by FastQC (v0.11.9) and removed PCR primmers by cutadaptor(v1.18). Trimmed data for ${treatment}_F1R1_F3R3.fq.gz and ${treatment}_F4R4.fq.gz were combined together. The combined data was mapped with ShapeMapper2 (v 2.1.3, parameters: --verbose --serial --nproc 16 --min-depth 1000). The RNA secondary structure was predicted by RNAfold (v 2.4.14, parameters: -p -d2 --shapeMethod=D).
Assembly: Target RNA sequence
Supplementary files format and content: TXT files include the read depths, standard errors and SHAPE reactivity of each base. Column "Norm_profile" is the SHAPE reactivity.

Submission date

Jan 03, 2023

Last update date

Jan 04, 2023

Contact name

Li Yang

E-mail(s)

[email protected]

Organization name

Fudan University

Department

Institutes of Biological Sciences