title: GSM3443055: NPAS4_OE_B1_R1; Mus musculus; RNA-Seq
library_construction_protocol: For ChIP of transcription factors and histone modifications, primary neurons (E16.5) were crosslinked via addition of 1% formaldehyde for 10 min at room temperature and quenched by the addition of 0.125 M glycine for 5 min at room temperature.  To ChIP the NCoR2 complex, we found it necessary to perform dual crosslinking by incubating neurons for 30 min with 1.5 mM EGS (ethylene glycol bis(succinimidyl succinate) (Fisher, 21565)), followed by 10 min incubation in 1% formaldehyde and standard quenching with 0.125 M glycine.  Nuclei were isolated from cell pellets by incubation in lysis buffer 1 (in mM: 100 HEPES-NaOH pH 7.5, 280 NaCl, 2 EDTA, 2 EGTA, 0.5% Triton X-100, 1% NP-40, 20% Glycerol) followed by washing in buffer containing 10 mM Tris-HCl pH 8.0, 200 mM NaCl.  Chromatin was sheared using a Bioruptor (Diagenode) on high power mode for 50 cycles with 30 sec pulses in sonication buffer (in mM: 10 Tris-HCl pH 8.0, 100 NaCl, 1 EDTA, 0.5 EGTA, 0.1% Na-Deoxycholate, 0.5% N-Lauroylsarcosine).  For H2K27ac ChIP, 10 mM sodium butyrate (Fisher NC9851678) was added to all buffers. Following sonication, chromatin was supplemented with 1% Triton and was incubated overnight with the following antibodies: H3K27ac (Abcam 4729, 0.2 μg for 2.5 million cells ), 5-8 μg Arnt2 (in house protein A purified anti-serum; see below), 8 μg Npas4 (custom made protein A purified anti-serum), NCoR2 (Millipore, 06891; 5 μg for 60-70 μg chromatin; Thermo PA1-843; 5 μg for 60-70 μg chromatin (40-50 million cells)); Tbl1 (Abcam 24528; 5 μg for 60-70 ug chromatin (40-50 million cells)); Hdac3 (Santa Cruz sc11416X; 5 μg for 60-70 μg chromatin (40-50 million cells). Libraries were generated using the Ovation Ultralow V2 kit according to the manufacturer's instructions and PCR amplified for 13-16 cycles depending on antibody. Library quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Seventy-five bp reads were generated on the Illumina Nextseq and subsequently analyzed with our standardized ChIP-seq data analysis pipeline (below). Tissue was collected in Trizol reagent and RNA was extracted using the RNAeasy Kit (Qiagen) according to the manufacturer's instructions. Total RNA (1000 ng) was used to generate libraries following rRNA depletion according to the manufacturer's instructions (NEBNext). Seventy-five bp reads were generated on the Illumina Nextseq and subsequently analyzed with our standardized RNA-seq data analysis pipeline (below). To assess regions of open chromatin, neurons were washed with cold PBS and lysed with 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal-630. 50,000 nuclei per condition were transposed using the Nextera DNA Library Prep Kit (Illumina).  Transposed libraries were amplified for 11 cycles and size selected for fragments ranging 200-1000 bp.