title: GSM6651817: M1-Left-11-Neuron-H3K27me3-R1; Mus musculus; ChIP-Seq
library_construction_protocol: Nuclei extraction from dissected brain tissue was performed using previously described protocol with minor modifications. All steps were conducted on ice and centrifugation was conducted at 4 ℃. A neocortex slice was thawed on ice and transferred to a glass tissue homogenizer (Sigma, d9036-1SET). 3 ml of ice-cold nuclei extraction buffer was added [0.32 M sucrose, 5mM CaCl2, 2 mM Mg(Ac)2 , 0.1 mM EDTA, 10 mM tris-HCl, 0.1% (v/v) Triton X-100, freshly added 1% protease inhibitor cocktail (PIC, Sigma-Aldrich), 0.1 mM PMSF, 1 mM DTT, and 0.06 U/µl ribonuclease (RNase) inhibitor (2313A,Takara Bio, needed when mRNA extraction was performed)]. The tissue was homogenized with 10 times of slow douncing using a loose pestle and 15 times of douncing using a tight pestle (D9063, Sigma-Aldrich). The homogenate was filtered through a 40-µm cell strainer (22363547, Thermo Fisher Scientific) into a 15-ml centrifuge tube and centrifuged at 1000 rcf for 10 min at 4 ℃. Supernatant was discarded and the pellet was resuspended in 0.5 ml of ice-cold nuclei extraction buffer. 0.75 ml of 50% (w/v) iodixanol (made by mixing 4 ml of OptiPrepTM gradient (Sigma-Aldrich) and 0.8 ml of diluent [150 mM KCl, 30 mM MgCl2, and 120 mM tris-HCl]) was added. The suspension was gently mixed by inverting the tube up and down 5 times and then centrifuged at 10,000 rcf for 20 min. Supernatant was carefully removed and the pellet incubated with 100 µl of 2% (w/v) normal goat serum (50062Z, Life Technologies) in Dulbecco's PBS (DPBS, Life technologies) on ice. The pellet was then resuspended and transferred into a new Eppendorf tube with an addition of 200 µl of 2% normal goat serum. To differentiate neurons from glia, 10 µl of 2 ng/µl Anti-NeuN antibody conjugated with Alexa 488 (MAB377X, EMD Millipore) was mixed with the suspension. The mixture was incubated at 4 ℃ for 1 h on a rotator. The nuclei solution was sorted using FACS (BS FACSAria, BD Bioscience) to separate neuronal (NeuN+) and glial (NeuN-) fractions.   Chromatin fragmentation:  M1-2: 4,000 nuclei per replicate was used so that both neuronal and glial fractions of every neocortex slice sample yielded enough material for two ChIP-seq replicates. The nuclei suspension (containing 8,000 nuclei) was topped up to 1 ml using Dulbecco's PBS (DPBS; 14190144, Life Technologies). 200 µl of 1.8M sucrose, 10 µl of 1M CaCl2, and 3 µl of 1M Mg(Ac)2 were then added sequentially. The sample was mixed gently by inverting the tube up and down and then incubated for 15 min on ice. The suspension was centrifuged at 3,000 rpm for 15 min at 4 ℃. Supernatant was carefully removed, and the pellet was resuspended in 10 µl DPBS. 0.1 µl of PIC, 0.1 µl of 100 mM PMSF and 10 µl of lysis buffer [4% Triton X-100, 100 mM tris (pH 7.5), 100 mM NaCl, and 30 mM MgCl2] were added, mixed by vortexing and then incubated at room temperature for 10 min. 1 µl of 0.1 M CaCl2 and 0.4 µl of 100 U/µl MNase (88216, Thermo Fisher Scientific) were then added, mixed by vortexing and incubated at room temperature for 10 min. 2.22 µl of 0.5 M EDTA (pH 8) was added to quench the rection. The sample was vortexed and incubated on ice for 10 min before it was centrifuged at 16,100 g for 5min. The supernatant (~20 µl, containing chromatin) was transferred to a new tube and 60 µl of IP buffer [20 mM Tris-HCl, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) sodium deoxycholate, 0.1% SDS, 1% (v/v) Triton X-100] was added to obtain a total volume of ~80 µl. The solution was finally split for two ChIP-seq replicates. The chromatin was stored on ice before loaded into the MOWChIP device.  M4-5: 200 µl of sorted neuronal nuclei suspension from one neocortex slice sample was used to prepare chromatin (containing ~30,000 nuclei). 2 µl of PIC, 2 µl of 100 mM PMSF and 200 µl of lysis buffer were added, mixed by vortexing and incubated at room temperature for 10 min. 4 µl of 1 M CaCl2 and 5 µl of 100 U MNase (88216, Thermo Fisher Scientific) were added, mixed by vortexing and incubated at room temperature for 10 min. 44 µl of 0.5 M EDTA was then added, mixed by vortexing and incubated on ice for 10 min. The mixture was finally centrifuged at 16,100g for 5 min at 4 °C. Supernatant containing fragmented chromatin (~ 440 µl) was collected into a new 1.5 ml tube and placed on ice before use. 70 µl of chromatin (containing chromatin from ~4,000 nuclei) was used for each MOWChIP-seq assay for consistency with data on M1-2. ChIP-seq libraries:  ~4,000 neuronal or glial nuclei were used to produce each ChIP-seq library and two technical replicates were generated on each neocortex slice sample. Libraries were constructed using 2S Plus DNA Library kit (10009877, IDT) and 2S CDI Adapter S1 indexing kit (10009895, IDT) following the manufacturer's instructions. 5% (v/v) EvaGreen (31000, Biotium) was added to the amplification mixture to monitor the progress of library amplification. Amplification was stopped once an increase of 2,000 RFU was reached. Library was eluted to 10 µl of low EDTA TE buffer.  mRNA-seq:  mRNA libraries were prepared following Smart-seq2 protocol55 with minor modifications. ~7,500 neuronal nuclei (from M3) were used to generate each RNA-seq library, and each slice sample has two technical replicates. 50 μl of neuronal suspension containing about 7,500 nuclei was used for RNA extraction using RNeasy Mini kit (QIAGEN) and RNase-Free DNase Set (79254, QIAGEN). Extracted RNA was concentrated using ethanol precipitation and resuspended in 5 μl of RNase-free water. 4.6 μl of RNA solution, 2 μl of 10 μM oligo-dT primer and 2 μl of 10 mM dNTP mix were mixed, incubated at 72°C for 3 mins and then stored on ice. 11.4 μl of reverse-transcription mix (made from 1 μl of SuperScript II reverse transcriptase (200 U/μl), 0.5 μL of RNase inhibitor (40 U/μl), 4 μL of Superscript II first-strand buffer, 1 μl of DTT (100mM), 4 μl of 5 M Betaine, 0.12 μl of 1 M MgCl2, 0.2 μl of TSO (10 μM), 0.58 μl of nuclease-free water) was added to the RNA solution and incubated at 42°C for 90 min, followed by 10 cycles of (50°C for 2 min, 42°C for 2 min), and at 70°C for 15 min. 25 μl KAPA HiFi HotStart ReadyMix, 0.5 μl of 10 μM IS PCR primers, 2.5 μl of 20x Evagreen, and 4 μl of RNase-free water were then added. cDNA was amplified by incubation at 98°C for 1 min, followed by 9-11 cycles (to achieve an increase of 50-100 RFU) of (98°C for 15 s, 67°C for 30 s, 72°C for 6 min). After amplification, cDNA was purified using 0.8× SPRIselect beads. Tn5 transposase (14-17 mg/ml) was produced following a published protocol56.  Tn5 transposase and T5/T7 transposons were assembled to form Tn5 transposome using a published protocol57. Briefly, 1.5 μl of 100 μM T5 transposon (IDT) was mixed with 1.5 μl of 100 μM pMENTS (a 5'-phosphorylated 19-bp mosaic end complementary oligonucleotide, IDT), while 1.5 μl of 100 μM T7 transposon (IDT) was mixed with 1.5 μl of 100 μM pMENTS. 3 μl of T5-pMENTS solution and 3 μl of T7-pMENTS solution were separately incubated at 95℃ for 5 min followed by a slow ramp to 25℃ at -0.1℃/sec. 2.5 μl of T5-pMENTS solution was then mixed with 2.5 μl of Tn5 transposase (14-17 mg/ml), while 2.5 μl of T7-pMENTS solution was mixed with 2.5 μl of Tn5 transposase. T5-Tn5 solution and T7-Tn5 solution were separately incubated at 37℃ for 1 h to form T5-Tn5 transposome and T7-Tn5 transposome, respectively. 4 μl of T5-Tn5 transposome and 4 μl of T7-Tn5 transposome were mixed to form 8 μl of assembled Tn5 transposome before it was mixed with 1 μl cDNA (containing ~500 pg cDNA) and 1 μl reaction buffer (TNP92110, Lucigen). The reaction mixture was incubated at 37 ℃ for 1 h. 1 μl of 10× stop solution (TNP92110, Lucigen) was added to the reaction mixture to stop tagmentation. Two rounds of 1× SPRIselect beads cleanup were performed to purify tagmented cDNA. The tagmented cDNA was then eluted in 10 μl low EDTA TE buffer. 25 μl KAPA HiFi HotStart ReadyMix was activated at 98 ℃ for 30 sec and then mixed with 9.5 μl tagmented cDNA and 10 μl Nuclease-free water, followed by incubation at 72 ℃ for 5 min. 1.5 μl of 25 M P5 primer, 1.5 μl of 25 M P7 primer and 2.5 μl 20x Evagreen were then added. The following amplification program was used to achieve a 300-400 RFU increase: 98 ℃ for 30 s; 11-12 cycles of (98 ℃ for 10 s, 63 ℃ for 30 s, 72 ℃ for 30 s) and 72 ℃ for 1 min. Amplified library was purified with 0.8× SPRIselect beads and eluted in 8 μl low EDTA TE buffer.