 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 22, 2024 |
| Title |
P21_brain_20um_RNA |
| Sample type |
SRA |
| |
|
| Source name |
Mouse brain
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Mouse brain
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The workflow of simultaneous profiling of chromatin accessibility or a panel of cell surface proteins with two histone modifications and gene expression on the same tissue cryosections is shown schematically in Fig. 1a and Extended Data Fig. 1a-b. For joint analysis of chromatin accessibility, two histone modifications, and transcriptome (Extended Data Fig. 1a, Methods), a frozen tissue section was first fixed with formaldehyde and in situ Tn5 transposition was performed to insert a unique barcoded ligation linker to the accessible DNA loci. The same tissue section was then incubated with two primary antibodies targeting different histone modifications simultaneously, such as the combination of H3K27me3 with H3K27ac or H3K4me3. Afterwards, species specific nanobody-Tn5 fusion proteins loaded with unique barcoded ligation linkers were added to enable the demultiplexing of different histone modification loci. Next, the tissue section was incubated with a biotinylated DNA adaptor containing a universal ligation linker and poly-T sequence that binds to the messenger RNA (mRNA) to perform in situ reverse transcription (RT). Next, barcodes A (A1-A50) and barcodes B (B1-B50) were sequentially flowed over the tissue using microchannels and were conjugated to the universal ligation liker via templated ligation, which formed a two-dimensional grid of spatially barcoded tissue pixels (n =2,500), allowing all of modalities from the same pixel share the same spatial barcodes. Finally, barcoded complementary DNA (cDNA) and genomic DNA (gDNA) fragments were released by reverse crosslinking. cDNAs were separated from gDNA by streptavidin beads. Sequencing libraries for cDNAs and gDNA were separately constructed. For co-profiling of protein, gene expression, and histone modifications (Extended Data Fig. 1b, Methods), the fixed frozen tissue section was first incubated with two primary antibodies targeting different histone modifications, followed by nanobody-Tn5 in situ transposition. The same tissue section was then labeled with a panel of barcoded poly-A-tailed oligo-conjugated antibodies, which recognize surface antigens. Next, in situ reverse transcript was performed using the Biotinylated poly-T RT primer to capture complimentary DNA of both poly-A-tailed oligo-conjugated antibodies and mRNA. The subsequent steps for spatial barcoding and library construction were similar to above discussed methods. The protein library and RNA library can be separated by SPRI bead purification.
Frozen tissue slides were thawed for 1 min at 37 °C. Tissue was fixed with formaldehyde (0.2%, with 0.05 U μl–1 RNase Inhibitor) for 5 min and quenched with 1.25 M glycine for a further 5 min. After fixation, tissue was washed twice with 1 ml of 0.5× DPBS-RI and cleaned with ddH2O. The sequential order for spatial multiple profiling is as follows: 1. ATAC-seq; 2. Nanobody-based CUT&Tag; 3. In situ reverse transcription; 4. Ligation of barcode A; 5. Ligation of barcode B; 6. Reverse crosslink; 7. gDNA and cDNA separation; 8. Library construction; 9. Library QC and sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
library strategy: spatial-RNA-seq
For ATAC and CUT&Tag data, linkers 1 and 2 are used for targeted filtering of read 2, aligning utilizing BWA followed by sorting and indexing via Samtools to facilitate efficient data handling and retrieval. This reformatting process assigned genome sequences to the first read and incorporated barcodes A and B into the second read. We aligned these fastq files against mouse (GRCm38) reference genomes. The conversion produced tsv-like fragments files, enriched with spatial and genomic information through the integration of barcode pairs, facilitating comprehensive downstream analysis. For RNA sequencing data, we refined read 2 to extract barcode A, barcode B, and the Unique Molecular Identifier (UMI). Using the Spatial Transcriptomics (ST) pipeline version 1.7.2, this processed data was mapped against the appropriate mouse (GRCm38) genome references. This step produced a gene matrix that captures both gene expression and spatial positioning data, encoded through the combination of barcodes A and B, enabling detailed spatial transcriptomic analysis.
Assembly: GRCm38
Supplementary files format and content: TSV files for downstream analysis. For CUT&Tag and ATAC-seq, the tsv file contains tissue location info (barcode A x barcode B) and fragments info on the genome.
Supplementary files format and content: For RNA-seq, Tab-delimited text files include counts for each pixels for each Sample.
Supplementary files format and content: The *spatial.tar.gz file is a compressed folder that stores spatial-related files including quality control (QC) images. These QC images are useful to confirm accurate fiducial alignment and tissue detection. Inside the folder, it contains tissue_lowres_image.png, scalefactors_json.json, and tissue_positions_list.csv. tissue_lowres_image.png is a downsampled version of the original, full-resolution light microscope image. scalefactors_json.json contains basic information about the pixel positions and sizes. tissue_positions_list.csv is a table with rows that correspond to pixels.
|
| |
|
| Submission date |
Apr 05, 2024 |
| Last update date |
Oct 22, 2024 |
| Contact name |
Liran Mao |
| Organization name |
University of Pennsylvania
|
| Department |
Perelman School of Medicine
|
| Street address |
422 Curie Boulevard
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE263333 |
Spatial-Mux-seq: Multiplexed spatial mapping of chromatin features, transcriptome, and proteins at tissue scale |
|
| Relations |
| BioSample |
SAMN40763510 |
| SRA |
SRX24172301 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8189712_P21_20um_RNA.tar.gz |
10.3 Mb |
(ftp)(http) |
TAR |
| GSM8189712_P21_20um_spatial_RNA.tar.gz |
743.7 Kb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
