|
| Status |
Public on Nov 16, 2021 |
| Title |
NIH-3T3 transfection 1x rep5 sRNA |
| Sample type |
SRA |
| |
|
| Source name |
NIH-3T3 transfected cells
|
| Organisms |
Mus musculus; synthetic construct |
| Characteristics |
cell type: NIH-3T3
treatment: 1x of the MapToCleave library
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For MapToCleave samples, total RNA and DNA were isolated using TRIzol reagent (Ambion). For validation samples, total RNA was extracted using a Zymo quick-RNA microprep kit (Zymo Research).
For MapToCleave samples, 1 ug of HEK-293T total RNA was used for standard small RNA library preparation using TruSeq small RNA kit v2 (Illumina). 1 ug of NIH-3T3 total RNA was used for preparation of small RNA libraries. 30 ng of the total RNA extracted from FACS-isolated MEF cells were used for preparation of small RNA libraries. The small RNA cDNA libraries were PCR-amplified in 15 cycles; the 75-bp single-end sequencing was carried out on a NextSeq500 (Illumina). DNA libraries were prepared in two steps: i) the DNA isolated from the HEK-293T and NIH-3T3 transfected cells was pre-amplified using custom primers compatible with Illumina TruSeq adapter sequences, ii) pre-amplified DNA fragments were multiplexed by PCR using Illumina TruSeq universal forward and single-indexed reverse PCR primers. The DNA libraries were resolved on a 6% Novex TBE gel (Invitrogen), and the 280 – 300 bp fraction was isolated. The 150-bp single-end sequencing was carried out using Illumina NextSeq500. For validation samples, RNA libraries were prepared using TruSeq small RNA kit v2 (Illumina).
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
count_table_of_MapToCleave_sequences_in_HEK_NIH3T3_MEF_HeLa_cells.txt
NIH3T3_R5_sh-lib_1x_1000ng_NOR.sRNA
|
| Data processing |
Basecalls were performed using bcl2fastq v2.17.1 with the option --barcode-mismatches 0
The small RNA sequencing data were quality control (QC) checked using miRTrace v 1.0.1 qc mode. Human (miRTrace option -s hsa) databases were used as references for HEK-293T and HeLa samples and mouse (-s mmu) databases were used as references for NIH-3T3 and MEF samples. Illumina 3’ adapter sequence TGGAATTCT was provided for 3’ adapter trimming.
For MapToCleave data, The QC qualified reads of each sample were then aligned to the prepared reference sequences, which contained the sequences from the MapToCleave library and the endogenous human (for HEK-293T and HeLa sample) or mouse (for NIH-3T3 and MEF sample) miRNA hairpin sequences that are excluded from the MapToCleave library but are included in miRbase v 21. The alignment was performed using bowtie v 1.1.2 without allowing mismatches (-v 0). The reads that were uniquely mapped to the forward strand of the reference sequences were considered for the hairpin expression measurement.
For validation data, the QC qualified reads were used to quantify expression of the human miRNA hairpins and the modified hairpins (see Supplementary Data 10 of the linked publication) using miRDeep2 v 2.0.0.8 quantifier.pl with allowing 0 mismatches (-g 0) and allowing multi-mapping. The human miRNA hairpin sequences downloaded from miRBase v21 and the modified hairpin sequences were used as the precursor reference (-p). The corresponding human miRNA sequences from miRBase v21 and the manually curated miRNA sequences of the modified hairpins (see Supplementary Data 10 of the linked publication) are used as the mature reference (-m). The hairpin sequence counts are measured by summing up the number of small RNA reads that mapped to an interval 2 nucleotides upstream and 5 nucleotides downstream of the 5p or/and 3p miRNA sequence from the hairpin.
Supplementary_files_format_and_content: tab-delimited text file includes raw counts of MapToCleave sequences for each sample.
Supplementary_files_format_and_content: tab-delimited text file includes raw counts and normalized counts of the modified hairpins and the endogenous human miRNA hairpins for each sample.
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| |
|
| Submission date |
Mar 16, 2021 |
| Last update date |
Nov 16, 2021 |
| Contact name |
Marc Riemer Friedländer |
| E-mail(s) |
marc.friedlander@scilifelab.se
|
| Organization name |
Stockholm University / SciLifeLab
|
| Department |
Department of Molecular Biosciences, The Wenner-Gren Institute
|
| Lab |
Lab Friedländer
|
| Street address |
Svante Arrhenius väg 20C
|
| City |
Stockholm |
| ZIP/Postal code |
10691 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL29860 |
| Series (1) |
| GSE169020 |
MapToCleave: high-throughput profiling of microRNA biogenesis in living cells |
|
| Relations |
| BioSample |
SAMN18320397 |
| SRA |
SRX10355559 |
