GSM6785865: tetOWapl_mCherry_omp_tta_F6_rep1; Mus musculus; RNA-Seq
For RNA-seq: RNA  was  isolated  from  tissue  using  TRIzol.  Cell  lysate  was  extracted  with  bromo-chloropropane and RNA was precipitated with 100% isopropanol supplemented with 10 μg of glycobluefor 10 min at room temperature and then pelleted at 16,000 x g for 30 min at  4°C.  The  RNA  pellet  was  washed  once  with  75%  ethanol  and  then  resuspended  in RNase-free water to a maximal concentration of 200 ng/μl. Genomic DNA contaminants were   removed   by   Turbo   DNase.   Removal   of   Turbo   DNase   was   performed   by phenol:chloroform  extraction  and  RNA  was  precipitated  as  described  above  and resuspended in RNase-free water and stored at -80°C.   For Raphe RNA-seq: RNA  was  isolated  from  coronal  sections  of  the  Raphe  (20  μm  thick)  obtainedfrom paraformaldehyde-fixed   brain   (described   in   more   detail   below,   see   paragraph “Preparation of Mouse Brain Tissue Sections”). After 3x 5 minutes washes of the slides with 1X PBS, a small portion of each section containing approximately the Raphe region was extracted. Then, the RNA was isolated using  RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Invitrogen) as instructed by the manufacturer.   For in situ HiC was carried out according to Rao et al. 2014.  For ChIP-seq: Cells were crosslinked with 1% formaldehyde for  10 minutes  at room  temperature. Formaldehyde  was  quenched  by  adding  glycine to  a  final concentration of 0.125 M for 5 minutes at room temperature. Cells were then washed with 1X cold PBS with protein inhibitors twice and pelleted. Cell pellets were stored at -80°C till use. Cells were lysed in lysis buffer (50 mM Tris pH 7.5, 140 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100) for 10 minutes. Nuclei were spun for 10 minutes at 1000 x g and resuspended in the sonication buffer (10 mM Tris pH 7.5, 0.5% SDS) as 5million nuclei per 300 μl sonication buffer. Chromatin was sheared by Covaris (Peak power 105.0; Duty Factor 2.0; Cycle/Burst 200; Treatment 960 sec; Temperature 4-8°C). Following a spin at 13,000 x g for 10 minutes to remove debris, the sheared chromatin was diluted such that the final binding buffer concentration was 15 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) and incubated for 2 hours with dynabeads G pre-equilibrated in the binding buffer for pre-clearing of the chromatin. Post-cleared chromatin was then incubated with the specific antibody overnight (1 μg of antibody was used per 5 million nuclei). The next day, dynabeads G were added to the chromatin-antibody mix for 2 hours. A total of four washes with 1X wash buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate) and one wash with TE buffer (10 mM Tris pH 7.5, 1 mM EDTA) were performed. The elution was performed at65°C for 1 hour in the elution buffer (1% SDS, 250 mM NaCl, 2 mM DTT).  All steps, with the exception of the elution, were performed at 4°C. All buffers, with the exception of the TE and elution buffer contained 1X protease inhibitors. The eluted chromatin was reverse-crosslinked overnight at 65°C and the DNA was purified with AMPure  XP  beads  (Beckman  Coulter). For RNA-Seq experiments, raw FASTQ files were aligned with either Tophat or STAR using hg19 or mm10 reference genomes. When libraries were made following the SMARTer Stranded Total RNA-Seq, the initial 4 base pairs of both paired reads were trimmed prior to alignment.   For single cell RNA-seq, raw FASTQ files were analyzed using cellranger.    For in situ Hi-C experiments, raw FASTQ files were analyzed using HiC-Pro and hic files were generated (Servant et al. 2015).