Illumina HiSeq 2500 sequencing; Nuclear SLIC-CAGE of human cortical neurons from Cornelia de Lange Syndrome patients and control individuals
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)