GSM2199918: S3_Neuronal_Nuclei; Homo sapiens; OTHER
50mg of frozen powdered 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. 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 FACSAria flow cytometer (BD Biosciences) equipped with a 70μm nozzle. Briefly, 50,000 sorted nuclei were centrifuged at 500 ×g for 10 min, 4°C. Pellets were re-suspended 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. Samples were incubated at 37°C for 30 min and then purified using the MinElute Reaction Cleanup kit (Qiagen Cat #28204), eluting in 10µl of buffer EB. Following purification, library fragments were amplified using the Nextera index kit (Illumina Cat #FC-121-1011), using the following cycling conditions: 72°C for 5 minutes, 98°C for 30 seconds, followed by thermocycling at 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute for a total of 5 cycles. In order to prevent saturation due to over-amplification, a 5µl aliquot was then removed and subjected to qPCR for 20 cycles to calculate the optimal number of cycles needed for the remaining 45 μL reaction. The additional number of cycles was determined as follows: (1) Plot linear Rn vs. Cycle (2) Calculate the # of cycle that is corresponded to 1⁄4 of maximum fluorescent intensity. In general, we found adding 4-6 cycles to this estimate yielded optimal ATACseq libraries, as determined by analysis on Bioanalyzer High Sensitivity DNA Chips (Agilent technologies Cat#5067-4626). Libraries were amplified for a total of 9–14 cycles. Following PCR, ATACseq libraries were resolved on 2% agarose gels supplemented with SYBR green. Fragments ranging in size from 100bp-1Kbp were excised and purified (Qiagen Minelute Gel Extraction Kit – Qiagen Cat#28604). Libraries were quantified by Qubit HS DNA kit (Life technologies) and by quantitiative PCR (KAPA Biosystems Cat#KK4873) prior to sequencing. Libraries were sequenced on Hi-Seq2500 (Illumina) obtaining 2x50 paired-end reads.
GEO
GSM2199918