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| Status |
Public on May 20, 2019 |
| Title |
HEK293T-N12-KO NAD-Capture 3 |
| Sample type |
SRA |
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| Source name |
HEK293T-N12-KO NAD-Capture
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
genotype/variation: Nudt12-KO (N12-KO)
enrichment: NAD-Capture
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| Treatment protocol |
No treatment
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| Growth protocol |
HEK293T cells were cultured under standard conditions with no modifications
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by TRIzol extraction, following the manufacturers recommended protocol
enrichment protocol: RiboMinus enrichment for non-ribosomal RNAs was performed as recommended by the kit manufacturer
NAD-Capture enrichment was performed using the NAD-RNA Capture protocol (Cahova et al., 2015) with minor modification. NAD-Capture was carried out with 100µg total RNAs treated with 10µl 4-pentyn-1-ol (Sigma-Aldrich) and 1.5U Adenosine diphosphate-ribosylcyclase (ADPRC) in 100µl reaction containing 50mM HEPES, 5mM MgCl2 (pH 7) and 40U RNasin® Ribonuclease Inhibitor (Promega) at 37oC 60 minutes. The reaction minus ADPRC was used as background control. The reaction was stopped with phenol/chloroform extraction and RNAs were precipitated with ethanol. The precipitated RNAs were subjected to copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction by incubating with 250µM biotin-PEG3-azide, freshly mixed 1mM CuSO4, 0.5mM THPTA, 2mM Sodium Ascorbate in 100µl reaction with 50mM HEPES, 5mM MgCl2 (pH7) and 40U RNasin® Ribonuclease Inhibitor (Promega) at 30oC for 30 minutes. Following extraction with phenol/chloroform and RNA precipitation, the NAD clicked RNAs were captures by binding to 20µl streptavidin magnet beads at room temperature with gentle rocking for 1 hour in 100µl binding buffer (1M NaCl, 10mM HEPES (pH7) and 5mM EDTA). The beads were washed three time with wash buffer (8mM Urea, 50mM Tris-HCl (pH7.4), 0.25% Triton-X100) and three washes with nuclease free H2O.
RiboMinus-selected RNAs were reverse transcribed and converted to libraries using the Illumina TruSeq RNA Sample Preparation Kit v2.
For NAD-Capture RNAs, samples were reverse transcribed into cDNA and synthesized into dsDNA with the SMARTer® Universal Low Input RNA Kit for Sequencing (Clontech Laboratories, Inc.) following the manufacturer’s protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
NAD-N12-KO-3
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| Data processing |
Illumina bcl2fastq2-v2.17.1.14 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and mapped to hg19 whole genome using TopHat v2.1.1 (with Bowtie 2.2.8.0) using default settings.
All samples were modeled using cuffdiff with options -p 6 --no-update-check and the UCSC hg19 genes.gtf file from the Illumina iGenomes site
Raw cuffdiff output files were loaded into the R package cummeRbund
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated by cuffdiff and output by cummeRbund
Genome_build: hg19
Supplementary_files_format_and_content: Gzipped, comma-separated-values (CSV) file containing isoform_ID row labels and sample replicate column labels. The values are FPKM.
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| Submission date |
Feb 19, 2018 |
| Last update date |
May 20, 2019 |
| Contact name |
Ronald P. Hart |
| E-mail(s) |
rhart@rutgers.edu
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| Phone |
848-445-1783
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| Organization name |
Rutgers University
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| Department |
Cell Biology & Neuroscience
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| Street address |
604 Allison Rd Rm B430
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| City |
Piscataway |
| State/province |
NJ |
| ZIP/Postal code |
08854 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
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| Relations |
| BioSample |
SAMN08564392 |
| SRA |
SRX3721455 |
| 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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