GSM5579470: MGE organoid from individual L9; Homo sapiens; ATAC-seq
50mg of frozen brain tissue was homogenized in cold lysis buffer (0.32M Sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM, EDTA, 10mM Tris-HCl, pH8, 1 mM DTT, 0.1% Triton X-100) and filtered through a 40µm cell strainer. The flow-through was underlaid with sucrose solution (1.8 M Sucrose, 3 mM Mg(Ace)2, 1 mM DTT, 10 mM Tris-HCl, pH8) and subjected to ultracentrifugation at 24,000 rpm for 1 hour at 4°C. Pellets were thoroughly resuspended in 500µl DPBS and incubated in BSA (final concentration 0.1%) and anti-NeuN antibody (1:1000, Alexa488 conjugated, Millipore) under rotation for 1 hour, at 4 °C, in the dark. Prior to FANS sorting, DAPI (Thermoscientific) was added to a final concentration of 1µg/ml. DAPI positive neuronal (NeuN+) and non-neuronal (NeuN-) nuclei were sorted into tubes pre-coated with 5%BSA using a BD-FACSAria flow cytometer (BD Biosciences) equipped with a 70μm nozzle. ATAC-seq reactions were performed using an established protocol (PMID25559105) with minor modifications. Media was removed and replaced with 200 µl of 1X Trypsin and 100mM trehalose. Tubes were incubated at 37°C for 10 minutes, after which dissociation was performed by gentle pipetting. Trypsin was neutralized by the addition of 200µl DMEM +10% FBS containing 0.5 µl turbo DNase and samples incubated for an additional 20 min at 37°C. Cells were then centrifuged at 300 g for 10 min at 4°C and pellets resuspended in 250µl PBS + 5% BSA. DAPI (Thermofisher) was added to the cells and viable (DAPI-) cells sorted on a FACSAria flow cytometer (BD Biosciences). Following FACS, cell concentrations and viability were confirmed using a Countess automated cell counter (Life technologies) and, where available, 75,000 cells were carried forward for the ATAC-seq reactions. Cells were centrifuged at 300 g for 10 min at 4°C and, following removal of the supernatant, pellets were resuspended in lysis buffer (10mM Tris-Cl pH7.4, 10mM NaCl, 3mM MgCl, 0.1% IGEPAL CA630) by pipetting 20 times. Following resuspension, samples were centrifuged at 500 g  for 10 min at 4°C. Pellets were resuspended in transposase reaction mix (25 μL 2x TD Buffer (Illumina Cat #FC-121-1030) 2.5 μL Tn5 Transposase (Illumina Cat #FC-121-1030) and 22.5 μL Nuclease Free H2O) on ice. Reactions were incubated at 37°C for 30 min before being purified using the MinElute Reaction Cleanup kit (Qiagen Cat #28204). Following purification, library fragments were amplified using the Nextera index kit (Illumina Cat #FC-121-1011) as previously described (PMID25559105). Library quality was assessed on a Tapestation instrument using D5000 ScreenTapes (Agilent technologies Cat# 5067-5588). Following amplification, libraries were resolved on 2% agarose gels and fragments ranging in size from 100-1000bp were excised and purified (Qiagen Minelute Gel Extraction Kit – Qiagen Cat#28604). Before sequencing, library fragment sizes estimated using Tapestation D5000 ScreenTapes (Agilent technologies Cat# 5067-5588) and libraries quantified by quantitative PCR (KAPA Biosystems Cat#KK4873) and). Finally, libraries were sequenced on the NovaSeq6000 platform (Illumina) obtaining 2x100 paired-end reads.