Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: The mouse line C57Bl/6 was 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 cerebellum was rapidly dissected and frozen on dry ice and stored at -80 ℃ until used for nuclei isolation. Cerebellum 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 transferred into an ultracentrifugation tube (355631, Beckman Coulter) and underlayered with 9.5 ml sucrose cushion (1.8 M sucrose, 3 mM Mg(Ac)2 and 10 mM Tris-HCl). The homogenate was centrifuged at 32,300 rpm (107,164g) for 2.5 h at 4 ℃ with 70 Ti rotor (Optima L-90K Ultracentrifuge, Beckman Counter). The spinning was slowly braked to avoid disturbing the nuclei pellet. All the supernatant was removed and the nuclei was gently resuspended in 0.4 ml 10% normal goat serum (50062Z, Life Technologies) and 1.6 ml Dulbecco's phosphate-buffered saline (14190144, Life Technologies). For RNA-seq, 3 μl 40 U/μl RNase inhibitor was added into nuclei suspension. The integrity and the number of nuclei were checked under the microscope. 32 μl 2 ng/μl anti-NeuN antibody conjugated with Alexa 488 (MAB377X, EMD Millipore) was incubated with the nuclei suspension (2 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. SurfaceChIP-seq library construction. The sequencing libraries were constructed using TruePLEX DNA-seq kit (Rubicon Genomics) or Accel-NGS 2S plus DNA library kit (Swift bioscience). GM12878 samples starting with ≥ 100 cells using DNA oligo linker were prepared using TruePLEX kit. The other samples are prepared using Accel-NGS 2S plus kit following instructions. 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. 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.