GSM6043248: mRNA-seq PFC Cocaine Rep2; Rattus norvegicus; RNA-Seq
Brain punches of PFC and NAc tissues were extracted and snap-frozen (at -30°C). Cryosections of approximately 500 microns were used to dissect PFC and NAc punches on a –20°C frozen stage. Total RNA was extracted using the TRIZOL protocol. csRNA-seq: csRNA-seq was performed as described previously (Duttke et al., 2019). Briefly, small RNAs of 15–60 nt were size selected from 0.3–1.0 microgram of total RNA by denaturing gel electrophoresis. A 10% input sample was taken aside, and the remainder enriched for 5'-capped RNAs. Monophosphorylated RNAs were selectively degraded by Terminator 5'-phosphate-dependent exonuclease (Lucigen). Subsequent 5' dephosphorylation by quickCIP (NEB) followed by decapping with RppH (NEB) augments Cap-specific 5' adapter ligation by T4 RNA ligase 1 (NEB)(Hetzel et al., 2016). Thermostable quickCIP was used instead of rSAP and hence the bead clean-up step was skipped before heat denaturation prior to the second round of CIP treatment. The 3' adapter was ligated using truncated T4 RNA ligase 2 (NEB) prior 3' repair to select against degraded RNA fragments. Following cDNA synthesis, libraries were amplified for 11–14 cycles and sequenced SE75 on the Illumina NextSeq 500 sequencer.   RNA-seq: RNA sequencing libraries were generated using the Illumina Stranded mRNA Prep (Illumina, San Diego, CA). Samples were processed following manufacturer's instructions. Resulting libraries were multiplexed and sequenced with 100 basepair (bp) Paired End reads (PE100) to a depth of approximately 25 million reads per sample on an Illumina NovaSeq 6000.  Hi-C: PFC tissue as pulverized in liquid nitrogen. The Arima-Hi-C kit was used to construct the Hi-C libraries (#A410231, Arima Genomics). Sequencing of the libraries was conducted on an Illumina Novaseq S4 instrument.   snATAC-seq: Nuclei were isolated from brain tissue as previously described (Corces et al., 2018). Briefly, frozen tissue was homogenized using a 2 ml glass dounce with 1 ml cold 1x Homogenization Buffer (HB). Cell suspension was filtered using a 70 µm Flowmi strainer (BAH136800070, Millipore Sigma) and centrifuged at 350g for 5 min at 4°C. Nuclei were isolated by iodixanol (D1556, Millipore Sigma) density gradient. The nuclei iodixanol solution (25%) was layered on top of 40% and 30% iodixanol solutions. Samples were centrifuged in a swinging bucket centrifuge at 3,000g for 20 min at 4°C. Nuclei were isolated from the 30%-40% interface. Library preparation targeting the capture of ~6000 nuclei was carried out as detailed in the Chromium Next GEM Single Cell ATAC v1.1 manual (10x Genomics). Library sequencing was performed using the Illumina NovaSeq.