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| Status |
Public on Apr 04, 2024 |
| Title |
Single cell methylation, RNA, and ATAC sequencing of amygdala brain tissue from inbred mouse strains |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
Methylation profiling by high throughput sequencing
Expression profiling by high throughput sequencing
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| Summary |
Knowing the genes involved in quantitative traits provides a critical entry point to understanding the biological bases of behavior, but there are very few examples where the pathway from genetic locus to behavioral change is known. Here we address a key step towards that goal by deploying a test that directly queries whether a gene mediates the effect of a quantitative trait locus (QTL). To explore the role of specific genes in fear behavior, we mapped three fear-related traits, tested fourteen genes at six QTLs, and identified six genes. Four genes, Lsamp, Ptprd, Nptx2 and Sh3gl, have known roles in synapse function; the fifth gene, Psip1, is a transcriptional co-activator not previously implicated in behavior; the sixth is a long non-coding RNA 4933413L06Rik with no known function. Single nucleus transcriptomic and epigenetic analyses implicated excitatory neurons as likely mediating the genetic effects. Surprisingly, variation in transcriptome and epigenetic modalities between inbred strains occurred preferentially in excitatory neurons, suggesting that genetic variation is more permissible in excitatory than inhibitory neuronal circuits. Our results open a bottleneck in using genetic mapping of QTLs to find novel biology underlying behavior and prompt a reconsideration of expected relationships between genetic and functional variation.
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| Overall design |
Adult male animals (Jackson Laboratories) were euthanized at 10-16 weeks old in an isoflurane chamber and decapitated. The brain was removed and the amygdala was microdissected, snap frozen in dry ice, and stored at -80oC until processing. Tissue from ~2 animals were combined into a single tube and considered a replicate, with 2 replicates per strain for snmC-seq2, snRNA-seq, and snATAC-seq experiments. snmC-seq2 on microdissected tissue as previously described (Luo et al 2018). We selected for a 75-25 enrichment of neuronal vs non-neuronal nuclei during FACS sorting using NeuN-488/DAPI counterstains. For snRNA-seq, single nuclei suspension and library generation were completed at the Cedars Sinai Applied Genomics, Computation and Translational Core and followed the 10X protocol for the Chromium Next GEM Automated Single Cell 3’ Library and Gel Bead Kit v3.1 (cat# PN-100014) as described except for the following modifications: Suspensions from cell nuclei were generated using the recommended method from the 10X scMultiome protocol (CG000375 Rev C) to lyse cells and obtain nuclei. Following single nuclei suspension generation, nuclei were counterstained for 7-AAD and NeuN-405 antibody (Novus Biologicals, 1:200) and sorted on a MACSQuant Tyto prior to GEM generation. We selected for a 75-25 split of NeuN+/7-AAD+ nuclei for neurons and NeuN-/7-AAD+ for non-neuronal nuclei respectively. We captured ~10,000 nuclei per genotype per region per replicate on a single 10X GEM chip. All downstream library preparation was done according to the 10X protocol (CG000286) and sequenced on a Novaseq 6000 with a target of ~40-50k reads per nucleus. For snATAC-seq, single nuclei suspension and library generation were completed at the Cedars Sinai AGCT core and followed the 10X protocol for Next GEM scATAC-Seq v1.1 (PN-1000175) as described except for the following modifications: Nuclei suspensions were generated using the recommended method from the 10X scMultiome protocol (CG000375 Rev C) to lyse cells and obtain nuclei Following single nuclei suspension generation, nuclei were counterstained for 7-AAD and sorted on a MACSQuant Tyto prior to GEM generation. NeuN was not used for neuronal enrichment due to dye incompatibility between our NeuN antibody and a nuclear counterstain. After the sort, we carried out permeabilization of nuclei as per the protocol. We aimed to capture 10,000 nuclei per well x 8 wells, for a total of 80,000 nuclei over 8 total samples (~10,000 nuclei per genotype per region per replicate). All downstream library preparation was done according to the 10X protocol (CG000209) and sequenced on a Novaseq 6000 with a target of >35k reads per nucleus.
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| Contributor(s) |
Chen PB, Flint J, Luo C |
| Citation(s) |
38697120 |
| BioProject |
PRJNA1030354 |
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| Submission date |
Mar 22, 2024 |
| Last update date |
May 30, 2024 |
| Contact name |
Patrick Chen |
| E-mail(s) |
pbc9@ucla.edu
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| Organization name |
UCLA
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| Department |
Psychiatry
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| Street address |
635 Charles E Young Dr S
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90024 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (24)
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| GSM8161200 |
snRNA-seq of mus musculus: adult male amydala DBA 1 [DBA_amy_1_RNA] |
| GSM8161201 |
snRNA-seq of mus musculus: adult male amydala DBA 2 [DBA_amy_2_RNA] |
| GSM8161202 |
snRNA-seq of mus musculus: adult male amydala BL6 1 [B6_amy_1_RNA] |
| GSM8161203 |
snRNA-seq of mus musculus: adult male amydala BL6 2 [B6_amy_2_RNA] |
| GSM8161204 |
snATAC-seq of mus musculus: adult male amygdala DBA 1 [DBA_amy_1_ATAC] |
| GSM8161205 |
snATAC-seq of mus musculus: adult male amygdala DBA 2 [DBA_amy_2_ATAC] |
| GSM8161206 |
snATAC-seq of mus musculus: adult male amygdala BL6 1 [B6_amy_1_ATAC] |
| GSM8161207 |
snATAC-seq of mus musculus: adult male amygdala BL6 2 [B6_amy_2_ATAC] |
| GSM8161208 |
snmC-seq2 of mus musculus: adult male amygdala DBA 1 [DBA_amy_1_methyl] |
| GSM8161209 |
snmC-seq2 of mus musculus: adult male amygdala DBA 2 [DBA_amy_2_methyl] |
| GSM8161210 |
snmC-seq2 of mus musculus: adult male amygdala BL6 1 [B6_amy_1_methyl] |
| GSM8161211 |
snmC-seq2 of mus musculus: adult male amygdala BL6 2 [B6_amy_2_methyl] |
| GSM8161212 |
snmC-seq2 of mus musculus: adult male amygdala AJ 1 [AJ_amy_1_methyl] |
| GSM8161213 |
snmC-seq2 of mus musculus: adult male amygdala AJ 2 [AJ_amy_2_methyl] |
| GSM8161214 |
snmC-seq2 of mus musculus: adult male amygdala FVB 1 [FVB_amy_1_methyl] |
| GSM8161215 |
snmC-seq2 of mus musculus: adult male amygdala FVB 2 [FVB_amy_2_methyl] |
| GSM8161216 |
snmC-seq2 of mus musculus: adult male amygdala BALBC 1 [BALBC_amy_1_methyl] |
| GSM8161217 |
snmC-seq2 of mus musculus: adult male amygdala BALBC 2 [BALBC_amy_2_methyl] |
| GSM8161218 |
snmC-seq2 of mus musculus: adult male amygdala CAST 1 [CAST_amy_1_methyl] |
| GSM8161219 |
snmC-seq2 of mus musculus: adult male amygdala CAST 2 [CAST_amy_2_methyl] |
| GSM8161220 |
snmC-seq2 of mus musculus: adult male amygdala PWK 1 [PWK_amy_1_methyl] |
| GSM8161221 |
snmC-seq2 of mus musculus: adult male amygdala PWK 2 [PWK_amy_2_methyl] |
| GSM8161222 |
snmC-seq2 of mus musculus: adult male amygdala WSB 1 [WSB_amy_1_methyl] |
| GSM8161223 |
snmC-seq2 of mus musculus: adult male amygdala WSB 2 [WSB_amy_2_methyl] |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSE262259_amy.L2.counts.ATAC.tsv.gz |
16.2 Mb |
(ftp)(http) |
TSV |
| GSE262259_amy.L2.counts.RNA.tsv.gz |
2.1 Mb |
(ftp)(http) |
TSV |
| GSE262259_amy.L2.metadata.ATAC.tsv.gz |
908 b |
(ftp)(http) |
TSV |
| GSE262259_amy.L2.metadata.RNA.tsv.gz |
960 b |
(ftp)(http) |
TSV |
| GSE262259_amy_allc_allstrains_alltypes_updatedcelltypes.tar.gz |
277.0 Gb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
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