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Sample GSM6833231

Query DataSets for GSM6833231

Status

Public on Jun 07, 2023

Title

28T-NBS

Sample type

SRA

Source name

breast

Organism

Homo sapiens

Characteristics

tissue: breast
cell type: breast cancer
treatment: untreated

Treatment protocol

Bisulfite treatment was carried out as reported by Khoddami et al. Briefly, samples were incubated with 312 µl of freshly prepared 5M sodium bisulfite (pH 5) and 3 µl of freshly prepared 100mM hydroquinone at 50°C, rotating for 16 h. Desulfonation was performed adding 0.5 ml of 2M Tris buffer pH 9.0 at 37°C for 2 h. After ethanol purification, samples were resuspended in 20 mM MgCl2 and 50 mM Tris HCl pH 7 and incubated at 75°C for 15’.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from frozen specimens of 34 breast tumors using the mirVana miRNA isolation kit (ThermoFisher Scientific). Human total RNA (Human XpressRef Universal Total RNA, Qiagen) was used as normal reference samples for analysis.
RNA library construction was performed with the TruSeq Stranded mRNA kit (Illumina) with some modifications. In particular, 100 ng of each beads-purified aqueous RNA samples were submitted to a second round of RNACleanXP beads purification to substitute water with the FPF (Fragment, Prime, Finish Mix) solution included in the library kit. Then the manufacturer’s protocol was followed. The Unique-dual indexes (Illumina) were used in the adapters ligation step. Each individual library was then quantified and quality-controlled using Qubit Fluorometer (ThermoFisher Scientific) and LabChip GX (Perkin Elmer). An unbalanced pooling design was used to achieve a ratio of 1 to 8 between untreated and BS-treated samples. After libraries unbalanced pooling, the final pool was quality checked again with Qubit, LabChip GX, and qPCR (KAPA and BIORAD).

Library strategy

Bisulfite-Seq

Library source

transcriptomic

Library selection

RANDOM

Instrument model

Illumina NovaSeq 6000

Data processing

Reads filtering (minimum base quality of Q30) and adapter trimming was performed with Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/).
Reads were aligned with meRanTK v1.2.1b (DOI: 10.1093/bioinformatics/btv647) using an index generated with the sequences of 5.8S, 18S, 28S, mt12S, and mt16S rRNAs obtained from the NCBI (https://www.ncbi.nlm.nih.gov/; #U13369.1:6623-6779, X03205.1, U13369.1:7935-12969, respectively.)). The meRanGs mode of meRanTK was used with default parameters.
The bisulfite-converted (RBS) and untreated samples (NBS) were then used as input for the ScorePseudouridinePosition tool of the RBSSeqTools v1.0 suite (23). For each RBS sample, the corresponding NBS sample was used as the non-bisulfite sample, using a threshold on the fraction of deletion reads at each position in the NBS sample of 10%. The per-position quantification and filtering produced by ScorePseudouridinePosition was used to analyze the fraction of modification of potential pseudouridine positions. The analysis on RBS samples consisted in filtering out positions not satisfying the following criteria: a) at least 10 reads in both RBS and NBS samples; b) at least 5 deletions in RBS; c) at least 5% of the reads with deletion on RBS samples; d) the percentage of deletion in NBS less than the threshold of 10%.
Assembly: hg38
Supplementary files format and content: tab-separated, fraction of modified reads at given position for each sample

Submission date

Dec 14, 2022

Last update date

Jun 07, 2023

Contact name

Erik Dassi

E-mail(s)

erik.dassi@unitn.it

Organization name

University of Trento

Department

CIBIO