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| Status |
Public on Jul 13, 2021 |
| Title |
EY1311 NAcc D1+ [YN1A] |
| Sample type |
SRA |
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| Source name |
Nucleus accumbens
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| Organism |
Mus musculus |
| Characteristics |
animal: EY1311
assay: cSNAIL ATAC-seq
surgery: headcap, retro-orbital AAV injection
virus: pAAV-Ef1a-DIO-Sun1-Gfp-WPRE-pA
genotype: Drd1-cre
project: Motor Learning ATAC-seq
cell type: D1+
age_week: 10
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| Treatment protocol |
For cSNAIL, cell type-specific ATAC-seq, we used the following treatment protocol on C57 mice. The cSNAIL genome (pAAV-Ef1a-DIO-Sun1-Gfp-WPRE-pA) contains loxP sites to invert the Sun1-Gfp fusion gene and integrate into the nuclear membrane of cells expressing the Cre gene, allowing these cell populations to be profiled for various genomic assays (Lawler et al, 2020 in press J. Neuro). We packaged the cSNAIL genome with AAV variant PHP.eB (pUCmini-iCAP-PHP.eB) in AAVpro(R) 293T cells (Takara, cat #632273). Viviana Gradinaru provided us with the pUCmini-iCAP-PHP.eB (http://n2t.net/addgene:103005; RRID: Addgene 103005)(Chan et al., 2017). We precipitated viral particles with polyethylene glycol, isolated with ultracentrifugation on an iodixanol density gradient, and purified in PBS with centrifugation washes and 0.2µM syringe filtration. We injected each mouse with 4.0 x 1011vg into the retro-orbital cavity under isoflurane anesthesia. We allowed the virus to incubate in the animal for 3-4 weeks to reach peak expression. We closely monitored the health of the animals throughout the length of the virus incubation and did not note any concerns.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We performed bulk and cSNAIL ATAC-seq from cortex (CTX) and dorsal striatum/nucleus accumbens C57Bl/6J mice as described in Buenrostro et al., 2015(Buenrostro et al., 2015) with the following minor differences in buffers and reagents. We euthanized mice with isoflurane, rapidly decapitated to extract the brain, and sectioned it in ice-cold oxygenated aCSF (119mM NaCl, 2.5 mM KCl, 1mM NaH2PO4(monobasic), 26.2mM NaHCO3, 11mM glucose) at 200-micron sections on a vibratome (Leica VT1200). We further micro-dissected sections for cortex and dorsal striatum on a stereo microscope and transferred dissected regions into chilled lysis buffer (Buenrostro et al., 2015). We dounce homogenized the dissected brains in 5mL of lysis buffer with the loose pestle (pestle A) in a 15mL glass dounce homogenizer (Pyrex #7722-15). We washed nuclei lysate off the pestle with 5mL of lysis buffer and filtered the nuclei through a 70-micron cell strainer into a 50mL conical tube. We washed the dounce homogenizer again with 10mL of BL buffer and transferred the lysate through the 70-micron filter (Foxx 1170C02). We pelleted the 20 mL of nuclei lysate at 2,000 x g for 10 minutes in a refrigerated centrifuge at 4°C. We discarded the supernatant and resuspended the nuclei in 100-300 microliters of water to approximate a concentration of 1-2 million nuclei/ mL. We filtered the nuclei suspension through a 40-micron cell strainer. We stained a sample of nuclei with DAPI (Invitrogen #D1206) and counted the sample to measure 50k nuclei per ATAC-seq transposition reaction. On the day of the ATAC-seq experiments, we dissected brain regions from fresh tissue and extracted nuclei in the same manner as described for bulk tissue experiments. Then, we sorted the nuclei suspension into Sun1GFP+ (Cre+) and Sun1GFP- (Cre-) fractions using affinity purification with Protein G Dynabeads (Thermo Fisher, cat. 10004D). A pre-clearing incubation with beads and nuclei for 10-15 minutes removes effects from non-specific binding events. Next, we incubated the remaining free nuclei with anti-GFP antibody (Invitrogen, #G10362) for 30 minutes to bind Sun1GFP. Finally, we added new beads to the solution to conjugate with the antibody and incubated the reaction for an additional 20 minutes. The pre-clear step and all incubations took place in wash buffer (0.25M Sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine with KOH to pH 7.8, and 0.4% IGEPAL) at 4°C with end-to-end rotation. After the binding process, we separated bead-bound nuclei on a magnet, washed three times with wash buffer, and filtered through a 20µM filter to ensure purity. We resuspended nuclei in nuclease-free water for input into the ATAC-seq tagmentation reaction. We performed nuclei quantification and tagmentation in the same manner described for bulk tissue ATAC-seq above. We list in the table below the number of animals, the genotypes, and which regions collected for ATAC-seq experiments in this study. N=2 Pvalb-cre samples from CPU/NAc region had received a sham surgery with saline injection into the external globus pallidus 5 days before they were sacrificed (Lawler et al, 2020 in press J. Neuro.) N=2 Drd1-cre samples from both CPU and NAc regions had received headcap surgeries 3 weeks before they were sacrificed. Both Pvalb-cre and Drd1-cre were overall healthy at time of sacrifice.
The remaining steps follow the Buenrostro et al., 2015(Buenrostro et al., 2015) protocol for tagmentation and library amplification.
We shallowly sequenced barcoded ATAC-seq libraries at 1-5 million reads per sample on an Illumina MiSeq and processed individual samples through the ENCODE pipeline for initial quality control. We used these QC measures (clear periodicity, library complexity, and minimal bottlenecking) to filter out low-quality samples and re-pooled a balanced library for paired-end deep sequencing on an Illumina NextSeq to target 30 million uniquely mapped fragments per sample after mitochondrial DNA and PCR duplicate removal. These raw sequencing files entered processing through the ENCODE ATAC-seq pipeline as above by merging technical replicates and grouping biological replicates by brain region for each pipeline run.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
this sample recieved headcap
YN1A
processed data file:
Pfenning_D1pos_Nac.narrowPeak.gz
cSNAIL_featureCounts_RangeSummarizedExperiment_n31_20200827.RDS
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| Data processing |
We processed raw FASTQ files of ATAC-seq experiments with the official ENCODE ATAC-seq pipeline (Landt et al., 2012) accessed by https://github.com/ENCODE-DCC/atac-seq-pipeline. We ran this pipeline using the mm10 genome assembly for mouse and the hg38 genome for human with the following settings: smooth_win = 150, multimapping = 0, idr_thresh = 0.1, cap_num_peak = 300,000, keep_irregular_chr_in_bfilt_peak = true. We grouped biological replicates when processing data to obtain individual de-duplicated, filtered bam files and reproducible (IDR) peaks for each condition. Unless otherwise stated, we used the optimal reproducible set of peaks for downstream analyses. We removed samples that had low periodicity indicated by ENCODE quality control metrics and reprocessed the remaining replicates with the pipeline.
We created a consensus set of non-overlapping IDR peaks from the ATAC-seq pipeline for cSNAIL ATAC-seq and Mo et al. INTACT samples (Tissue: Ctx, Cpu, and NAc ; Celltype: EXC, PV, SST, VIP, D1, D2). We extended the peak set 200bp up- and down-stream, count overlapping fragments with Rsubread v2.0.1 using the de-duplicated BAM files from the pipeline(Liao et al., 2014). This is the raw counts matrix including experiments published with this work and
We used Deeptools v3.5.0 to convert the deduplicated BAM files to normalized bigWig files (Ramírez et al., 2016).
Genome_build: mm10
Supplementary_files_format_and_content: peak: optimal IDR peaks from ENCODE pipeline. bigWig = normalized bigWig files from BAM alignment. counts= featurecounts summarized rangedExperiment of consensus IDR peaks across celltypes/tissues
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| Submission date |
Nov 12, 2020 |
| Last update date |
Jul 13, 2021 |
| Contact name |
BaDoi Nguyen Phan |
| E-mail(s) |
badoi.phan@pitt.edu
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| Organization name |
Carnegie Mellon University
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| Department |
Computational Biology
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| Lab |
Pfenning Lab
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| Street address |
5000 Forbes Ave
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15213 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE161374 |
Addiction-associated genetic variants implicate brain cell type- and region-specific cis-regulatory elements in addiction neurobiology |
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| Relations |
| BioSample |
SAMN16784788 |
| SRA |
SRX9498793 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4905606_YN1A_NAcc_D1.bw |
144.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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