title: GSM5741319: OmpGFP_WT_RNASeq_Rep1; Mus musculus; RNA-Seq
library_construction_protocol: 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 ChiP-Seq experiments, raw FASTQ files were aligned using Bowtie2 using hg19 reference genome upon adapter sequences removal using CutAdapt. Uniquely aligning reads were selected using Samtools and reads with alignment quality below 30 (-q 30) were removed. The HOMER software package was used to generate signal tracks.   For in situ Hi-C experiments, raw FASTQ files were analyzed using HiC-Pro and hic files were generated (Servant et al. 2015).