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| Status |
Public on Sep 07, 2022 |
| Title |
TTseq_GM10253A_ExoKD_2 |
| Sample type |
SRA |
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| Source name |
Somatic cell hybrid
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| Organisms |
Homo sapiens; Cricetulus griseus |
| Characteristics |
cell type: GM10253A
treatment: Exosome siRNA 48hr
molecule subtype: nascent RNA
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| Treatment protocol |
Nascent RNA was labelled by adding 500 μM 4-thiouridine (4sU)(Sigma, T4509) to untreated and siRNA (exosome) treated GM10253A and HybNeo3 cells and incubating at 37°C for 10 min. For siRNA treatment, cells (GM10253A and HybNeo3; 10%–20% confluent) were transfected with 10 nM Silencer Select Pre-designed siRNA targeting EXOSC3 (Ambion, Life Technologies) using Lipofectamine RNAi MAX (ThermoFisher) 24 h after seeding and again 48 h later. After a further 48 h exosome knockdown was confirmed by western blotting and TTseq was performed. Silencer Select RNA sequence for EXOSC3 were GAGATATATTCAAAGTTGA, part number s83102. The control RNA was Stealth RNAi siRNA Negative Control (ThermoFisher).
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| Growth protocol |
Human/Hamster hybrid cell lines GM10253A and HybNeo3 were grown in RPMI 1640 with L-Glutamine, 10% FCS and penicillin and streptomycin. Human parenral lymphoblastoid cell line was grown in RPMI 1640 with L-Glutamine, 20% FCS and penicillin and streptomycin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed with TRIzol (Invitrogen) following the manufacturers’ instructions. After DNase treatment (Turbo DNase, Thermo Fisher Scientific) RNA concentration and purity were determined using a NanoDrop. RNA (70 μg) was fragmented in 100 μl H20 to <1.5kb by 20 cycles of 30 seconds on/30 seconds off at high power in a Biorupter plus and RNA size assessed by agarose gel electrophoresis. Fragmented 4sU labelled RNA was biotinylated by adding 140 μl of EZ-Link Biotin-HPDP (1mg.ml-1 in dimethylformamide; Pierce, 21341), 70 μl of 10 x biotinylation buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA) and H20 to a final volume of 700 μl. This was incubated at room temperature for 1.5 h with rotation. Unincorporated biotin-HPDP was removed by two rounds of chloroform extraction with 2 ml Phase lock gel heavy tubes (Eppendorf). RNA was precipitated with 1/10 volume of 5M NaCl and an equal volume of Isopropanol. This was inverted to mix and incubated at room temperature for 10 min followed by centrifugation at 10,000g for 20 min at room temperature. RNA pellet was washed with 80% EtOH and centrifuged at 13,000 rpm for 10 min at 4°C. RNA was resuspended in 100 μl H20 and dissolved by heating to 40°C for 10 min with agitation. RNA was then immediately placed on ice and RNA concentration determined by Nanodrop spectrophotometer. Biotinylated 4sU labelled RNAs were then recovered using μMACS Streptavidin MicroBeads (Miltenyi, 130-074-101) and separation on a μMACS Separator. For the concentration of total RNA in μg per sample an equal amount in μl of Streptavidin microbeads was added. This was incubated at room temperature for 15 min with rotation. μMacs columns were equilibrated with 900 μl room temperature washing buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween20). The RNA/streptavidin bead solution was then applied to the column followed by three washes with 900 μl of washing buffer at 65°C and three washes with 900 μl of washing buffer at room temperature. RNA was eluted with 2 x 100 μl of fresh elution buffer (100 mM dithiothreitol in RNase-free H20 ) directly into 2 ml lobind tubes (Eppendorf) containing 700μl Buffer RLT (RNeasy MinElute Cleanup Kit, Qiagen). 500 μl of 100% ethanol was added to the RNA solution, and mixed thoroughly by pipetting before RNA was purified through RNAeasy MinElute Spin Columns. RNA concentration was determined using a Nanodrop.
Libraries for RNA-seq were prepared and indexed using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB #E7645L) and NEBNext Singleplex Oligos for Illumina (NEB #E7335, E7500) following the manufacturer’s instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Chinese hamster/human hybrid retaining human chromosome #3
TTseq_GM10253A_ExoKD_chr3_200.bw
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| Data processing |
50bp paired end reads had adapters removed (Trim Galore) and were aligned to a Human(hg38) /Hamster (GCA_003668045.1) hybrid reference genome using Bowtie2 and processed with Samtools v1.6, and the deepTools “bamCoverage” tool with RPKM normalisation across Human chromsome 3 and with a 200bp bin size.
Genome_build: Human(hg38) /Hamster (GCA_003668045.1) hybrid reference genome
Supplementary_files_format_and_content: Bigwig files of combined replicates for human chromosome 3 only,with RPKM normalisation and binsize of 200bp.
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| Submission date |
Feb 04, 2022 |
| Last update date |
Sep 07, 2022 |
| Contact name |
Catherine Naughton |
| E-mail(s) |
[email protected]
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| Organization name |
University of Edinburgh
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| Department |
MRC Human Genetics Unit
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| Lab |
Nick Gilbert Lab
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| Street address |
Crewe Road
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| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
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| Platform ID |
GPL31910 |
| Series (2) |
| GSE195886 |
Human centromere repositioning activates transcription and opens chromatin fibre structure |
| GSE196155 |
Human centromere repositioning activates transcription and opens chromatin fibre structure [TTseq] |
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| Relations |
| BioSample |
SAMN25653734 |
| SRA |
SRX14045522 |
| 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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