|
| Status |
Public on Feb 28, 2021 |
| Title |
healthy control NeuN-positive Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
cortical neuronal nuclei
|
| Organism |
Homo sapiens |
| Characteristics |
disorder: healthy control
tissue: NeuN-positive
|
| Treatment protocol |
Isolation of nuclei was performed as previously described (Krishnaswami et al., 2016) with minor modifications. Briefly, 50-250mg of pre-frontal cortical grey matter was homogenised in homogenisation buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 1μM DTT, 1X Proteinase Inhibitor w/o EDTA (Roche), 0.4U μl-1 RNaseIn (ThermoFisher) 0.2U μl-1 Superasin (ThermoFisher), 1μM DAPI) and centrifuged through an iodixanol gradient (Sigma). Pelleted nuclei were washed and then stained with NeuN antibody (Abcam, ab190195) in staining buffer (PBS, 1%BSA, 0.2U/μl RNaseIn (Thermofisher), NeuN antibody (1:200)) for one hour at 4oC. For negative controls antibody was excluded. Nuclei were then sorted on BD Fusion, and collected in wash buffer (PBS, 1%BSA, 0.2U/μl RNaseIn (Thermofisher)), before proceeding to downstream RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the PicoPure RNA isolation kit (ThermoFisher) following the manufacturer's instructions.
40ng of total RNA was used to prepare libraries using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (ribozero ribosomal RNA depletion), following the manufacturer recommendations. Library quality and quantity were assessed on a Bionalayser and Qubit respectively. Libraries were sequenced on an Illumina Hiseq2500 (v4 chemistry) and at least 40million paired end 100bp reads per sample were generated per library..
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
RNA-seq of healthy control cortical neuronal nuceli
Ctrl_NueNngeVSNueNpos_GeneCounts.csv
1739NeuNpos
|
| Data processing |
Illumina CASAVA 1.8.4 software used for basecalling.
Reads were mapped to hg38 whole genome using tophat2 (2.0.11) with argument “--library-type fr-firststrand"
Gene annotations were obtained from iGenome UCSC hg38. Gene-based read count were obtained using featureCounts function from Rsubread Bioconductor package
Genome_build: hg38
Supplementary_files_format_and_content: csv file includes Gene counts for each Sample
|
| |
|
| Submission date |
May 08, 2020 |
| Last update date |
Feb 28, 2021 |
| Contact name |
LMS Bioinformatics Core |
| E-mail(s) |
bioinformatics@lms.mrc.ac.uk
|
| Organization name |
MRC London Institute of Medical Sciences
|
| Department |
Bioinformatics Core
|
| Street address |
Hammersmith Hospital Campus, Du Cane Road
|
| City |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE150121 |
Human cortical nuclei RNAseq |
| GSE150130 |
Partial rescue of neuronal genes deregulated in CdLS by cohesin |
|
| Relations |
| BioSample |
SAMN14854894 |
| SRA |
SRX8293770 |
