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| Status |
Public on Jun 19, 2024 |
| Title |
EU_seq_shCDK12_rep1 |
| Sample type |
SRA |
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| Source name |
U2OS
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| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
treatment: shCDK12
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was then extracted and purified using the Quick-RNATM Miniprep Kit (Zymo Research, cat. #R1055) according to the manufacturer’s instructions. Nascent RNA was biotinylated and purified starting from 50 μg of total RNA. EU was coupled to a cleavable biotin-azide linker (Azide-PEG(3+3)-S-S-biotin) (kind gift from Thanos Halazonetis) using the Click-IT chemistry as shown below: 50 μg of RNA (40 μg of human EU-labelled RNA and 10 μg of murine EU-labelled RNA) were mixed with the same volume of 2X Click-IT reaction cocktail (171 mM Tris-HCl pH 8, 8 mM CuSO4, 200 mM sodium-L-ascorbate, 0,1 mM biotin–azide) and incubated for 30 minutes at RT with gentle shaking. RNA was precipitated and the EU-labelled RNA was then isolated using 50 μl of Dynabeads MyOne streptavidin C1 (Invitrogen, Cat. No. 65001) each sample, that were washed before use two times with Binding and Washing Buffer 1X (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.5% Tween-20), followed by two washes with solution A (0.1 M NaOH, 0.05 M NaCl) and two washes with solution B (0.1 M NaCl). After washing, the beads were resuspended to twice the original volume with Binding and Washing Buffer 2X. RNase OUT (1:100, Invitrogen, Cat. No. 10777-019) was added to RNA and then samples were heated at 70°C for 5 minutes and placed back on ice to remove secondary structures. Then EU-labelled RNA was mixed with an equal volume of washed beads (100 µL) and incubated for 30 minutes on a rotating wheel at room temperature. The RNA-beads complex was then washed three times with Binding and Washing Buffer 1X and once with RNase free-water. Finally, the EU-labelled RNA was eluted by incubating the streptavidin beads with 55 μl of 2% β -mercaptoethanol (Calbiochem, Cat. No. 444203) in 10 mM Tris-HCl pH. 8 for 1 h at room temperature. 5 μl of each sample were used for Qubit quantification (RNA HS assay kit; Thermofisher, Cat. No. Q32852).
TruSeq Stranded Total RNA Gold (Illumina, Cat. No. 20020598)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Read filtering with fastx toolkit 0.0.13.2: fastq_masker -Q33 -q 20 -r N -v
reads alignment with STAR v. 2.7.5c
Quantifying read counts over features with featureCounts v. 1.4.5-p1: featureCounts -T 2 -p -P -a gtffile
Assembly: hg19
Supplementary files format and content: count_matrix.xls: tab-delimited text file containing raw counts for each gene
Supplementary files format and content: EU_seq_shNT_OHT_rep3.bw: bigwig of EU_seq_shNT_OHT_rep3 sample
Supplementary files format and content: EU_seq_shCDK12_OHT_rep3.bw: bigwig of EU_seq_shCDK12_OHT_rep3 sample
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| Submission date |
Jul 05, 2023 |
| Last update date |
Jun 19, 2024 |
| Contact name |
Stefano Campaner |
| E-mail(s) |
stefano.campaner@iit.it
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| Organization name |
fondazione Istituto italiano di tecnologia
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| Department |
Center for Genomic Science
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| Lab |
Cancer Biology
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| Street address |
Via adamello 16
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| City |
Milano |
| ZIP/Postal code |
20139 |
| Country |
Italy |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE236549 |
CDK12 prevents MYC-induced transcription-replication conflicts [EU-seq] |
| GSE236553 |
CDK12 prevents MYC-induced transcription-replication conflicts |
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| Relations |
| BioSample |
SAMN36313372 |
| SRA |
SRX20904043 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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