description: Protocols: 50-250 mg of pre-frontal cortex grey matter were obtained from:1) National Institutes of Health - NIH NeuroBiobank2) Edinburgh - The Edinburgh Brain Bank, Edinburgh University3) CHOP - Children's Hospital of Philadelphia, Dr. Ian Krantz(the origin of each sample is marked under \provider\ attribute) 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, collected in wash buffer (PBS, 1%BSA, 0.2U/μl RNaseIn (Thermofisher)), and pelleted before proceeding with downstream RNA extraction. RNA was extracted from nuclei using Picopure RNA Isolation Kit (Thermofisher) according to manufacturer's instructions. Residual DNA was eliminated using RNAse-Free DNase Set (Qiagen). About 50 ng of nuclear RNA from each sample was used for SLIC-CAGE TSS mapping. Briefly, 4.95 μg of the carrier RNA mix were added to each sample prior to reverse transcription, followed by the cap-trapping steps designed to isolate capped RNA polymerase II transcripts. The carrier was degraded from the final library prior to sequencing using homing endonucleases. The target library derived from the neuronal nuclear RNA was PCR-amplified and purified using AMPure beads (Beckman Coulter) to remove short PCR artifacts (< 200bp, size selection using 0.8 x AMPure beads to sample ratio)
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-03-01T12:39:45Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-05-11T15:31:44Z
attributes.tag: External Id
attributes.value: SAMEA6823860
attributes.tag: INSDC center name
attributes.value: Institute of Clinical Sciences, Faculty of Medicine, Imperial College London and MRC London Institute of Medical Sciences
attributes.tag: INSDC first public
attributes.value: 2021-03-01T12:39:45Z
attributes.tag: INSDC last update
attributes.value: 2020-05-11T15:31:44Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9045:CDL-380P
attributes.tag: age
attributes.value: 19
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: Cornelia de Lange syndrome
attributes.tag: genotype
attributes.value: NIPBL 2965_2966insA
attributes.tag: individual
attributes.value: CDL-380P
attributes.tag: organism part
attributes.value: prefrontal cortex
attributes.tag: sample name
attributes.value: E-MTAB-9045:CDL-380P
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: male