 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 07, 2020 |
| Title |
RNABL30921_ADHD_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Whole blood
|
| Organism |
Homo sapiens |
| Characteristics |
condition: ADHD
twin pair id: 14
tissue: Whole blood
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with the Qiagen PAXgene blood RNA kit (P/N 762164) using a QIAcube automation system.
Total RNA, rRNA depleted
Ribosomal RNA depletion of approximately 0.5-2 ug of total RNA was performed using the Illumina Ribo-Zero Gold kit (#MRZG12324). First strand cDNA synthesis was carried out by initially incubating 7 µL of sample plus water (~30-50 ng RNA) at 95°C for 5 minutes and chilling on ice for 2 minutes. While on ice, 5 µL of 50 ng/µL random hexamers (Invitrogen #51709) and 1 µL of 10 mM dNTP (Invitrogen #Y02256) mix were added to the RNA and the mixture was incubated at 65°C for 5 minutes. While on ice, 4 µL of 5x buffer and 1 µL of 0.1 M DTT were mixed into the solution and incubated at 15°C for 20 minutes. Finally, 1 µL of RNase Inhibitor and 1 µL of 200 U/µL Superscript III Reverse Transcriptase (Invitrogen #18080-044) were added. The following program was run on the thermal cycler: 25°C for 10 minutes, 40°C for 40 minutes, 55°C for 50 minutes, and 85°C for 5 minutes. Upon completion, 1 µL each of 2 U/µL RNase H (Invitrogen #18021-071) and 50 U/µL RNase If (NEB #MO243) were used to digest the RNA strand at 37°C for 30 minutes and subsequently purified using EdgeBio Performa DTR Gel filtration cartridges (#42453). 3’ Poly A tailing was initiated by addition of 4 µL of 10x TdT buffer and 4 µL of 2.5 mM CoCl2 and water to 10 µL of cDNA, denatured at 95 °C for 5 minutes, and snap cooled. Then 250x dATP and 1 µL of 20 U/µL (diluted to 10 U/µL) Terminal Transferase (NEB #M0315) were added and the solution was incubated for 30 minutes at 37°C. Finally, 2 µL of 200 µM ddATP were spiked into the reaction which was incubated at 37°C for 1 hour, and 70°C for 10 minutes to inactivate the enzyme.
PCR-free RNA-Seq
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Helicos HeliScope |
| |
|
| Data processing |
Raw files were processed with Helisphere software package ver. 1.2.740
Reads aligned to the human genome with indexDPgenomic and filtered with parameters: min_len=25, min_score=4.3, best_only=1, global_ambiguity=all
Raw expression was calculated with a custom script: +1 is added if a read occurs inside an exon and +0.5 if read covers exonic junction
Raw counts were RPKM normalized with a custom script
Genome_build: GRCh38
Supplementary_files_format_and_content: TXT tab-separated file with DGE counts
|
| |
|
| Submission date |
Oct 06, 2020 |
| Last update date |
Oct 07, 2020 |
| Contact name |
Joel T. Nigg |
| Organization name |
Oregon Health & Science University
|
| Department |
Department of Psychiatry
|
| Lab |
Division of Psychology
|
| Street address |
3181 S.W. Sam Jackson Park Road
|
| City |
Protland |
| State/province |
Oregon |
| ZIP/Postal code |
97239-3098 |
| Country |
USA |
| |
|
| Platform ID |
GPL14761 |
| Series (1) |
| GSE159104 |
Biomarker Discovery in Attention Deficit Hyperactivity Disorder: RNA sequencing of Whole Blood in Discordant Twin and Case-Controlled Cohorts |
|
| Relations |
| BioSample |
SAMN16380359 |
| Supplementary data files not provided |
| Processed data are available on Series record |
| Raw data not provided for this record |
|
|
|
|
 |
