GSM2843601: Clozapine5_R1_RRBS; Mus musculus; Bisulfite-Seq
The mouse lines C57Bl/6 and 126S6/SvEv weres bred and maintained in groups of 3-4 per cage with food ad libitum. 8-10 week old mice were anesthetized with isoflurane and decapitated. The tissue was rapidly dissected and frozen on dry ice and stored at -80 ℃ until used for nuclei isolation.  The tissue was placed in 5 ml ice-cold nuclei extraction buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 0.1 mM EDTA, 10 mM Tris-HCl and 0.1% Triton X-100) with freshly added 50 μl protease inhibitor cocktail (P8340, Sigma-Aldrich), 5 μl 100 mM PMSF and 5 μl 1 M DTT. In addition, 7.5 μl 40 U/μl RNase inhibitor (N2611, Promega) was also added when RNA-seq was conducted. Once thawed, the tissue was homogenized by slowly doucing for 15 times with a loose pestle (D9063, Sigma-Aldrich) and 25 times with a tight pestle (D9063, Sigma-Aldrich). The homogenate was filtered with 40 µm nylon mesh cell strainer (22363547, Fisher Scientific) and then transferred into a 15 ml centrifugation tube. The sample was centrifuged at 1000 g for 10 min. The supernatant was removed and the cell pellet was gently resuspended in 1 ml cold nuclei extraction buffer with freshly added 10 μl protease inhibitor cocktail, 1 μl of 100 mM PMSF and 1 μl of 1 M DTT. 1.5 μl of 40 U/μl RNase inhibitor was mixed with the nuclei extraction buffer when RNA-seq was conducted. The 1 ml sample suspension was split into two 1.5-ml micro-centrifuge tubes (500 μl in each tube). Each sample (500 μl) was gently mixed with 0.75 ml of 50% iodixanol to yield 30% iodixanol solution. 50% iodixanol was prepared by adding 0.4 ml diluent (150 mM KCl, 30 mM MgCl2, and 120 mM Tris-HCl, pH 7.8) to 2 ml of 60% iodixanol (D1556, Sigma). The samples were then centrifuged at 10,000 g for 20 min before the supernatant was removed. The nuclei in each tube were incubated on ice for 10 min in 0.5 ml Dulbecco's phosphate-buffered saline (14190144, Life Technologies) containing 2.0% normal goat serum (50062Z, Life Technologies). The nuclei were resuspended and then pooled together (generating ~1 ml in total). For RNA-seq, 3 μl of 40 U/μl RNase inhibitor was added into the nuclei suspension. The integrity and the number of nuclei were checked under the microscope. 32 μl of 2 ng/μl anti-NeuN antibody conjugated with Alexa 488 (MAB377X, EMD Millipore) was incubated with the nuclei suspension (~1 ml) for 1 h at 4 ℃ on an end-to-end rotator. The stained samples were sorted using a FACS (BD FACSAria, BD Biosciences) with 50,000 to 100,000 unlabeled nuclei used as non-staining control. Human genomic DNA was purified from GM 12878 cells using QIAamp DNA Blood Mini Kit (Qiagen) following the manufacturer's protocol and suspended in 50 µl EB buffer. Mouse genomic DNA was purified using QIAamp DNA Blood Midi Kit (Qiagen) from nuclei and suspended in 200 µl AE buffer (10 mM Tris-Cl, 0.5 mM EDTA, pH 9.0). The eluate was reloaded on the column membrane and spun to maximize DNA yield. Mouse DNA was concentrated by ethanol precipitation to 20 µl EB buffer. RNA was extracted from ~10,000 nuclei using RNeasy Mini Kit (Qiagen). DNase treatment step was included following kit instructions to remove gDNA contamination. RRBS library construction. 5 µl genomic DNA (0.2-6 ng/µl) was digested by adding 1 µl of 20 U/µl MspI enzyme (R0106S, NEB), 2 µl NEB buffer2 and 12 µl H2O at 37 ℃ for 1 h and inactivated at 80 ℃ for 20 min. The fragment size was examined using 2.5% agarose gel (16550100, Thermo Fisher) in 0.5× TBE buffer (15581044, Thermo Fisher). The 3' terminal of digested DNA was end-repaired and an extra A which was required by adapter ligation was added on both strands in a single reaction. 1 µl of 5 U/µl Klenow exo- enzyme (M0212M, NEB) and 1.1 µl dNTP mixture (10 mM dATP, 1 mM dGTP and 1 mM dCTP, N0446S, NEB) were added into 20 µl digested sample. Ten-fold excess of dATP was added to increase A-tailing efficiency. The sample was incubated at 30 ℃ for 20 min (end-repair), 37 ℃ for 20 min (A-tailing) and 75 ℃ for 20 min (enzyme inactivation). The DNA (22.1 µl) was then purified by phenol–chloroform extraction and ethanol precipitation. DNA pellet was then resuspended in 17 μl EB buffer. 16.4 µl sample was transferred to a new 200 µl PCR tube. 1 µl of 2000 U/µl T4 ligase (M0202T, NEB), 2 µl of supplied 10× T4 ligase buffer, 0.6 µl NEXTflex bisulfite-seq barcode (1.6-6 µM) were mixed with the sample for ligation. The ligation reaction was carried out at 16 ℃ for 16 h and followed by 65 ℃ for 10 min for heat inactivation. The heating of the thermal cycler lid was turned off to protect the T4 ligase. Gel-free procedure 17, 28 instead of gel cutting was used for RRBS library construction. Ligated DNA (20 µl) was purified using AMPure XP beads with bead suspension to sample volumetric ratio of 1.5: 1. The DNA was eluted from Ampure beads by 20 µl EB buffer. The elute was concentrated by ethanol precipitation and then suspended in 0.7 µl to facilitate loading into the microfluidic device. The converted DNA was amplifed by PCR (13 cycles for 10 ng, and 16-18 cycles for 1-0.3 ng). After PCR amplification, a double size selection using AMPure XP beads was performed. Large DNA fragments were removed by adding 30 μl AMPure bead suspension and incubating for 5 min. The beads that had large DNA bound were discarded and the supernatant was preserved. 30 μl AMPure bead suspension were then added into the supernatant and incubated for 5 min. The supernatant was discarded and DNA bound to beads was eluted into 7 μl EB buffer.mRNA-seq library construction. RNA was extracted from ~100,000 nuclei using RNeasy Mini Kit (Qiagen). DNase treatment was included following manufacturer instructions to remove gDNA contamination. The RNA quality was detected by High Sensitivity RNA ScreenTape System (Agilent) to yield RNA integrity number (RIN) > 6. mRNA-seq libraries were prepared using SMART-seq v4 Ultra Low Input RNA kit (Clontech) and Nextera XT DNA library prep kit (Illumina) following instructions.
GEO Accession
GSM2843601