title: GSM5666952: wt12_ctx_e13_S42_R1_001; Mus musculus; RNA-Seq
library_construction_protocol: PROTOCOL 1: Intact nuclei were isolated by pipetting up and down twenty times in ice cold 0.5 mL Buffer 1 (300mM sucrose, 60mM KCl, 15mM NaCl, 15mM Tris-HCl, pH 7.5, 5mM MgCl2, 0.1mM EGTA, 1mM DTT, 1.1mM PMSF, Protease inhibitors), and then lysed on ice for 10 minutes after adding 0.5 mL Buffer 2 (300mM sucrose, 60mM KCl, 15mM NaCl, 15mM Tris-HCl, pH 7.5, 5mM MgCl2, 0.1mM EGTA, 0.1% NP-40, 1mM DTT, 1.1mM PMSF, Protease inhibitors). During these ten minutes, nuclei were counted using trypan blue and 50,000 nuclei were spun down at 7,000rpm for ten minutes at 4C. PROTOCOL 1: Nuclei were resuspended in 25uL Tagmentation buffer, 22.5 uL Nuclease Free H20, and 2.5 uL Tagmentation Enzyme from Nextera DNA Library Prep Kit, gently mixed, and placed in 37C water bath for thirty minutes. The tagmentation reaction was stopped by MinElute PCR purification and DNA was eluted in 10uL Nuclease Free water. ATAC-seq library generation was performed using Illumina barcode oligos as described (Buenrostro et al 2015), for 8-11 cycles PCR using NEBNext High Fidelity 2x PCR master mix. The number of cycles was empirically determined for each library by qPCR. Libraries were bioanalyzed using Agilent High Sensitivity DNA Kit, pooled together and sequenced on Hiseq 2500. PROTOCOL 2: Intact nuclei were isolated by pipetting up and down twenty times in ice cold 0.5 mL Buffer 1 (300mM sucrose, 60mM KCl, 15mM NaCl, 15mM Tris-HCl, pH 7.5, 5mM MgCl2, 0.1mM EGTA, 1mM DTT, 1.1mM PMSF, 50mM Sodium Butyrate, EDTA-free Protease inhibitors), and then lysed on ice for 10 minutes after adding 0.5 mL Buffer 2 (300mM sucrose, 60mM KCl, 15mM NaCl, 15mM Tris-HCl, pH 7.5, 5mM MgCl2, 0.1mM EGTA, 0.1% NP-40, 1mM DTT, 1.1mM PMSF, 50mM Sodium Butyrate,  EDTA-free Protease inhibitors). During this ten minutes, nuclei were counted using trypan blue and 500,000 nuclei were spun down at 7,000rpm for ten minutes at 4C.  Nuclei were resuspended in 0.250mL MNase buffer  (320mM sucrose, 50mM Tris-HCl, pH 7.5, 4mM MgCl2, 1mM CaCl2, 1.1mM PMSF, 50mM Sodium Butyrate) and incubated in a 37C water bath with 2 microliters Micrococcal Nuclease enzyme (NEB) for eight minutes.  Micrococcal Nuclease digestion was stopped by adding 10 microliters 0.5M EDTA, and chromatin was spun down for 10 minutes 10,000rpm 4C.  Soluble fraction “S1” supernatant was saved at 4C overnight, and “S2” fraction was dialyzed overnight in 250uL dialysis buffer at 4C (1mm Tris-HCl pH 7.5, 0.2mM EDTA, 0.1mM PMSF, 50mM Sodium Butyrate, Protease Inhibitors).  Next day S1 and S2 fractions were combined, 50 microliters were saved as input, and Chromatin immunoprecipitation was set up in ChIP buffer: 50mM Tris, pH 7.5, 10mM EDTA, 125 mM NaCl1, 0.1% Tween. 250m M Sodium Butyrate was supplemented for H3K27ac ChIPs.  1 microliter of antibody was added to 1mL chromatin in ChIP buffer and incubated overnight at 4C rotating.  Protein A and Protein G beads (10 microliters for each ChIP) were blocked overnight in 700uL ChIP buffer, 20 uL yeast tRNA (20mg/mL), and 300uL BSA (10mg/mL). Beads were washed three times for five minutes on ice in Wash buffer 1 (50 mM Tris, pH 7.5 , 10mM EDTA , 125mM NaCl, 0.1% Tween-20, with protease inhibitors and 5mM sodium butyrate) and three times in Wash buffer 2 (50 mM Tris, pH 7.5 , 10mM EDTA , 175mM NaCl, 0.1% NP-40, with protease inhibitors and 5mM sodium butyrate), and ChIP DNA was eluted in elution buffer at 37C and purified by phenol chloroform extraction and ethanol precipitation.  PROTOCOL 2: Sequencing libraries were made using Nugen Ovation Ultralow V2 kit and quantified by Agilent High Sensitivity DNA kit on the Agilent bioanalyzer. PROTOCOL 3: From each dissection, RNA was isolated using QIAGEN RNeasy mini columns. RNA was treated with Turbo DNAse and inactivated.  RNA quality was assessed using Agilent Bioanalyzer RNA Nano kit, and samples with RNA Integrity Number values greater than 9.0 were used for subsequent profiling. PROTOCOL 3: RNA-seq libraries were generated using Nugen's Ovation Mouse RNA-seq kit and amplified for 15 cycles.  Libraries were quantified using Agilent Bioanalyzer High Sensitivity DNA kit, and sequenced on Hiseq 2500 using paired end sequencing. PROTOCOL 4: After spinning down nuclei at 7,000rpm for ten minutes at 4C, we methodically followed the protocol for CUT &RUN (Skene, Henikoff, and Henikoff 2018), starting at step 6 “Resuspend in 1mL of wash buffer at RT by gentle pipetting”. Antibodies were used at the following dilutions: POGZ 1:500, ADNP 1:13, Hp1g 1:500, IgG 1:1000. For the final DNA extraction step, we performed phenol-chloroform extraction and ethanol precipitation. PROTOCOL 4: We generated libraries using Nugen's Ovation Ultralow V2 kit, and amplified libraries for 16 cycles, and bioanalyzed libraries using the Agilent High Sensitivity DNA kit and sequenced libraries on the Hiseq 2500.