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Sample GSM8224455

Query DataSets for GSM8224455

Status

Public on Oct 03, 2024

Title

Structure-seq, HEK293T, Control, NAI, Rep2

Sample type

SRA

Source name

HEK293T

Organism

Homo sapiens

Characteristics

cell line: HEK293T
genotype: WT
treatment: DMSO+NAI

Treatment protocol

For HEK293T cells, the control and STM2457-treated cells were harvested in 1X PBS buffer (Thermo Fisher Scientific, 10010049) and probed with NAI (50 mM in final) or 5% DMSO as negative control for 15 min at 37°C, 500 rpm. Then DTT (200mM in final) was added immediately to quench each probing reaction. After spinning down the cells (~ 500 g) for 3 min at room temperature, total RNAs were extracted and quantified using RNeasy Plus Mini Kit (Qiagen, 74136) and Nanodrop ND-1000 Spectrophotometer.

Extracted molecule

polyA RNA

Extraction protocol

poly(A)-selected RNAs
Each library was prepared with 500 ng poly(A)-selected RNAs after 2 rounds of selection (Thermo Fisher Scientific, AM1922). The poly(A) RNAs were fragmented to around 300 nt length at 95°C for 1 min and purified using RNA clean and concentrator (Zymo research, R1016). The fragmented RNA was then subjected to a 5' dephosphorylation reaction at 37°C for 30 min by adding rSAP (NEB, M0371L) and PNK enzyme (NEB, M0201S) in 1× T4 PNK buffer (NEB, B0201S). Next, the 3’adapter was ligated to the dephosphorylated RNA using T4 RNA ligase 2 KQ (NEB, M0373L) at 25°C for 1 h. Excess adapters were digested and removed by adding RecJf (NEB, M0331) and 5’ deadenylase (NEB, M0331) for 30 min at 30°C followed by column purification (Zymo research, R1016). The following reverse transcription (RT) was performed in 1 X RT buffer (20 mM Tris, pH 7.5, 4 mM MgCl2, 1 mM DTT, 0.5 mM dNTPs, 150 mM LiCl) with SuperScript III enzyme (Thermo Scientific, 18080093) at 42°C for 40 min. RNA was digested by 2M NaOH and the reaction was neutralized with 1M Tris-HCl (pH 7.5) followed by column purification (Zymo research, R1016). The 5’adapter (for C2C12 libraries) or dU adapter (for HEK293T libraries) was ligated to the cDNA with Quick T4 DNA ligase (NEB, M2200L) and incubated in 37°C overnight. For the C2C12 library, the ligated product was run on the 10% denaturing urea-TBE acrylamide gel (Thermo Scientific, EC68752BOX), and the 100–400 nt region was cut and dissolved in 1X TEN250 buffer (1X TE 7.4, 0.25 M NaCl) at 80°C for 30 min followed by column purification (Zymo research, R1016). For the HEK293T libraries, the ligated product was first purified by column (Zymo research, R1016), then USER II enzyme (NEB, M5509) was added to cleave the product followed by a second column purification (Zymo research, R1016). The purified product was used for PCR cycle test and amplification with different barcoded reverse primer. PCR products with 150–400 bp were then cut and purified with 2% agarose gel and column (Zymo research, D4008).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq X Plus

Description

HEK293T_control_statistic_reactivity.csv

Data processing

The adapters were first removed using Cutadapt v1.15 and the reads shorter than 20nt or with quality score lower than 30 were filtered out.
The remaining reads were mapped to human or mouse transcriptome reference using Bowtie2 v2.3.3.1.
Next, the mapped reads with more than 3 mismatches or mismatch at the first base were removed, RT stops were counted and reactivity scores were calculated using StructureFold2 package.
Assembly: hg38
Supplementary files format and content: comma-delimited text file includes SHAPE reactivity score of each transcript
Library strategy: Structure-seq

Submission date

Apr 23, 2024

Last update date

Oct 03, 2024

Contact name

Yuwei ZHANG

E-mail(s)

[email protected]

Phone

95390159

Organization name

The Chinese University of Hong Kong