Instrument: Illumina HiSeq 4000
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: RANDOM
Layout: PAIRED
Construction protocol: A detailed protocol for MethylC-seq libraries preparation can be found in Urich et al., (2015). MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing. Nat Protoc 10, 475-483. Nuclei isolation from mouse and human cortical tissues was performed as described in (Lister et al., 2013) with the following modifications: Proteinase inhibitor (11836153001, Roche) and RNAse Inhibitor (30 U/ml, PRN2611 from Promega) was added to the lysis buffer and sucrose gradients. After centrifugation, nuclei were resupended in 0.5% BSA (AM2616, Ambion) PBS (Ca2+ and Mg2+ free, 14190-144 from Life Technologies,) with protein and RNAse inhibitors. Isolated nuclei from mouse and human tissues were labeled by incubation with 1:1000 dilution of AlexaFluor488 conjugated anti-NeuN antibody (MAB377X, Millipore) at 4°C for 1 hour. Nuclei isolated from CLSun1-G35-Cre line were incubated with AlexaFluor647 conjugated anti-NeuN antibody (anti-NeuN antibody MAB377 labeled using Apex Alexa Fluor 647. A10475, Life Technologies) and AlexaFluor488 conjugated anti-GFP antibody (A21311, Life Technologies). Fluorescence activated sorting of single nuclei was performed using a BD Influx sorter with an 85 µm nozzle at 22.5 PSI sheath pressure. Single nuclei were sorted into each well of a 384-well plate preloaded with 2 µl of Proteinase K digestion buffer (1 µl M-Digestion Buffer, 0.1 µl 20 µg/µl Proteinase K and 0.9 µl H2O). The alignment of receiving 384-well plate was performed by sorting sheath flow into wells of an empty plate and making adjustment based on the liquid drop position. Single cell (1 drop single) mode was selected to ensure the stringency of sorting. Steps of library preparation prior to SPRI purification were performed in a horizontal laminar flow hood to minimize environmental DNA contamination. Bisulfite conversion of single nuclei was carried out using Zymo EZ-96 DNA Methylation-DirectTM Kit (Deep Well Format, cat. #D5023) following the product’s manual with reduced reaction volume. 384-well plate (ThermoFisher Armadillo PCR Plate cat. # AB2384) containing FACS isolated single nuclei were heated at 50°C for 20 min. 25 µl CT Conversion Reagent was added to each well followed by pipetting up-and-down to mix. Plates were treated with a following program using a thermocycler: 98°C for 8 min, 64°C for 3.5 hours and 4°C. Each well of Zymo-Spin™ I-96 Binding Plates was preloaded with 150 µl M-binding buffer. Bisulfite conversion reactions were transferred from 384-well plates to I-96 Binding Plates followed by pipetting up-and-down to mix. I-96 Binding Plates were centrifuged at 5,000g for 5 min. Wells were washed with 400 µl of M-Wash Buffer followed by centrifugation at 5,000g for 5 min. 200 µl of M-Desulphonation Buffer was added to each well and was incubated for 15 min at room temperature before removed by centrifugation at 5,000g for 5 min. Each well was then washed with 400 µl of M-Wash Buffer for two rounds. 12 µl of M-Elution Buffer was added to each well and was incubated for 5 min at room temperature. I-96 Binding Plate was placed above a 96-well PCR plate (Applied Biosystems MicroAmp® EnduraPlateTM cat. # 4483348) and was centrifuged at 5,000g for 3min. 9 µl of eluted DNA was commonly collected in each well of the PCR plate. 1 µl of 5 µM P5L-AD002-N9, P5L-AD006-N9, P5L-AD008-N9 or P5L-AD010-N9 indexed random primer was added to each well of 96-well plate followed by mixing with vortexing. All DNA oligos were purchased from Integrated DNA Technologies (IDT). Plate was heated at 95°C for 3 min to denature the sample and was immediately chilled on ice for more than 2 min. 10 µl enzyme mix containing 2 µl of Blue Buffer (Enzymatics cat. # B0110), 1 µl of 10mM dNTP (NEB cat. # N0447L), 1 µl of Klenow exo- (50U/µl, Enzymatics cat. # P7010-HC-L) and 6 µl H2O. After mixing with vortexing, plate was treated with a following program: 4°C for 5 min, ramp up to 25°C at 0.1°C/sec, 25°C for 5 min, ramp up to 37°C at 0.1°C/sec, 37°C for 60 min, 4°C. 2 µl of Exonuclease 1 (20U/µl, Enzymatics cat. # X8010L) was added to each well followed by mixing with vortexing. Plate was incubated at 37°C for 30 min and then 4°C. 17.6 µl of home-made SPRI beads was added to each well. Sample/bead mixture from four plates indexed using distinct indexed random primers were combined followed by pipetting up-and-down to mix. Sample/bead mixture was incubated at room temperature for 5 min before placed on a 96-well magnetic separator. Supernatant was removed from each well followed by three rounds of washing with 180 µl of 80% ethanol. After air drying beads at room temperature, 10 µl M-Elution buffer was added to each well to fully resuspend the beads. Eluted sample was transferred to a new 96-well PCR plate. PCR plate was heated at 95°C for 3 min to denature the sample and was immediately chilled on ice for more than 2 min. 10.5 µl Adaptase (Swift Biosciences) mix (2 µl Buffer G1, 2 µl Reagent G2, 1.25 µl Reagent G3, 0.5 µl Enzyme G5, 0.5 µl Enzyme G6 and 4.25 µl M-Elution buffer) was added into each well followed by mixing with vortexing. Plates was incubated at 37°C at 30 min and then 4°C. 30 µl PCR mix (25 µl KAPA HiFi HotStart ReadyMix, KAPA BIOSYSTEMS, cat. # KK2602, 1 µl 30 µM P5 indexing primer and 5 µl 10 µM P7 indexing primer) was added into each well followed by mixing with vortexing. PCR plate was treated with the following program: 95 °C for 2 min, 98°C for 30 sec, 17 cycles of (98°C for 15 sec, 64°C for 30 sec, 72°C for 2min), 72°C for 5 min then 4°C. PCR products were cleaned up using 0.8x SPRI beads and was combined into one tube for each 96–well plate. Pooled PCR product was resolved on 2% agarose gel, smear between 400 bp and 2 Kb were excised and purified using QIAquick Gel Extraction Kit (Qiagen cat. # 28706). Library concentration was determined using Qubit® dsDNA HS (High Sensitivity) Assay Kit (Invitrogen cat. # Q32851).