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Sample GSM8039506

Query DataSets for GSM8039506

Status

Public on Jan 31, 2024

Title

Denatured, biological replicate 1

Sample type

SRA

Source name

In vitro

Organism

Homo sapiens

Characteristics

cell type: In vitro
treatment: 1M7

Treatment protocol

For each sample, 5 pmol of RNA mixture of pri-miRNAs and positive control RNAs (each pri-miRNA : each positive control RNA = 1 : 2) was refolded in 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM MgCl2 in a final volume of 10 μL at 37℃ for 20 min. After folding, RNAs were modified in the presence of 10 mM 1M7 and incubated at 37℃ for 75 sec. The “No-reagent” control (Untreated), containing DMSO rather than SHAPE reagent, was performed in parallel. For the denatured sample, RNAs were modified using 1M7 (final 10 mM) under strongly denaturing conditions in 50 mM HEPES pH 8.0, 4 mM EDTA and 50% formamide at 95℃ for 1 min.

Growth protocol

The DNA constructs were pooled, purified (QIAquick PCR Purification Kit, QIAGEN), and treated with SurveyorⓇ Mutation Detection Kit (IDT) to remove DNA with mutations. Then, the DNAs were gel-purified and amplified by PCR using KAPA HiFi HotStart ReadyMix (Kapa Biosystems) with primers; 5′-TAATACGACTCACTATAGGGCCTATTCAGTTACAGCG-3′ (forward) and 5′-m(2′-O-Me)GmUTGCTAGCTTCAGTACG-3′ (reverse). Finally, pri-miRNAs were prepared by in vitro transcription using MEGAscript™ T7 Transcription Kit (Ambion).

Extracted molecule

other

Extraction protocol

After the modification, RNAs were precipitated by ethanol.
the SHAPE-modified RNAs were subjected to reverse transcription for 3 h at 42℃ using SuperScript II (Invitrogen). The RT primer (5′-CCTTGGCACCCGAGAATTCCANNNNNNGTTGCTAGCTTCAGTACG-3′) contains the sequence complementary to the 3′ custom adapter, which is followed by six random nucleotides and RNA 5′ adapter sequence of TruSeq Small RNA Library Preparation Kit (Illumina). Reverse transcriptase buffer contained 0.5 mM premixed dNTPs, 50 mM Tris-HCl pH 8.0, 75 mM KCl, 6 mM MnCl2 and 10 mM DTT.
After reverse transcription, cDNAs were purified by Agencourt RNAClean XP (Beckman). Sequencing libraries were generated using the one-step PCR approach. The PCR reactions were performed using Q5 Hot Start High-Fidelity DNA polymerase (NEB) with the forward PCR primer (5′-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATCNNNNNNGCCTATTCAGTTACAGCG-3′), which contains the P5 sequence for Illumina paired-end sequencing at the 5′ end, six random nucleotides, and the sequence complementary to the 5′ common sequence. RNA PCR Index Primers of Truseq Small RNA Library Preparation Kit were used as reverse primers. One-step PCR was performed for 3 cycles to tag cDNAs and generate the final library for sequencing. PCR products were purified using AMPure XP beads (Beckman). The library was sequenced on Illumina Hiseq X-10 (150x150 paired-end run) with 50% of the PhiX control library (Illumina).

Library strategy

OTHER

Library source

other

Library selection

other

Instrument model

BGISEQ-500

Description

SHAPE_reactivity.csv
SHAPE-based_pri-miRNA_structures.csv

Data processing

Raw sequences were quality-filtered using the FASTX Toolkit (v. 0.0.13.2; http://hannonlab.cshl.edu/fastx_toolkit; fastq_quality_filter -q 25 -p 80). The filtered sequences were aligned to pri-miRNA construct sequences using bowtie2 with reduced mismatch and gap extension penalties (version 2.2.479; --end-to-end --mp 5,1 --rdg 5,1 --rfg 5,1 --no-mixed --no-unal --norc).
From the aligned sequences, mutations at each nucleotide position were counted using the ShapeMapper package (version 1.0). Ambiguously mapped reads were discarded, and consecutive mutations were merged during alignment parsing (parseAlignment -combine_strand -deletion_masking -remove_ambig_del -min_map_qual 2).
SHAPE reactivity was calculated by dividing the difference between mutation rates in the 1M7-treated and 1M7-untreated samples by mutation rates in the RNA-denatured sample, as described in the previous study. SHAPE reactivities were normalized by the 90th percentile of reactivities within each RNA species.
Secondary structures were modeled using the RNAstructure package (Fold, version 5.841).
Assembly: Hg38
Assembly: Pri-miRNA construct sequences (Table S2)
Supplementary files format and content: SHAPE reactivities and secondary structures were prepared as csv files.
Library strategy: SHAPE-Seq

Submission date

Jan 28, 2024

Last update date

Jan 31, 2024

Contact name

Seungchan Baek

E-mail(s)

[email protected]

Phone

8573969069

Organization name

Broad Institute