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| Status |
Public on Oct 12, 2023 |
| Title |
Control |
| Sample type |
SRA |
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| Source name |
Brain
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| Organism |
Mus musculus |
| Characteristics |
tissue: Brain
strain: C57
genotype: WT
treatment: Saline
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| Treatment protocol |
C02KL was administered with ketamine intraperitoneally (i.p.) at a dosage of 10 mg/kg. Other mice were administered with saline.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mice were euthanized using CO2. Brain tissues were dissected out and embedded in Tissue-Tek (OCT) and snap-frozen using an isopentane–dry ice slurry. The OCT-embedded brains were cryosectioned sagittally to a thickness of 10 mm and placed onto spatially barcoded arrays on ST glass slides (10x Genomics).
Briefly, glass slides with tissue sections attached to spatially barcoded arrays were permeabilized by collagenase in HBSS-BSA buffer for 20 min and 0.1% pepsin in 0.1M HCl for 5 min at 37℃. Then reverse transcription was performed by adding the cDNA synthesis master mix at 42℃ overnight to get the cDNA attached on the array. Tissue on the array was then degraded by incubation with 2.5 mg/mL proteinase K in PDK buffer at 56℃ for 1 h with intermittent shaking at 300 rpm, which left the cDNA coupled to the arrayed oligonucleotides on the slide. Then the probe was collected by adding 70 μL of release mix to the array chambers. The release mix was composed of 100 U/mL USER enzyme, 1× first strand buffer, 0.5 mM dNTPs, and 0.19 μg/μL BSA. Incubation was performed at 37℃ for 2 h, and 65 μL of the released probe mixture was collected from each array chamber at the end of the incubation. Then the library of the released probes was prepared through second strand cDNA synthesis, in vitro transcription, adaptor ligation, second cDNA synthesis, qPCR quantification, and PCR amplification. Second strand cDNA synthesis was performed by adding 5 mL mixture to each sample. The mixture was composed of 2.7× First Strand Buffer, 3.7 U/μL DNA polymerase I, and 0.18 U/μL RNaseH and the reaction was incubated at 16℃ for 2 h. Then, 5 mL of T4 DNA polymerase were added to each sample and the samples were incubated at 16℃ for 20 min. In vitro transcription was performed by adding 5.6 μL mixture to each sample and incubating the samples at 37℃ for 14 h. The mixture was composed of 1× T7 Reaction Buffer, 7.5 mM of each NTP, 1× T7 Enzyme Mix, and 1 U/mL SUPERaseIN. Adaptor ligation was performed by adding 4.5 μL of the ligation mixture to each sample and incubating the samples at 25℃ for 1 h. The ligation mixture was obtained by incubating 8 μL of amplified RNA and 2.5 μL of ligation adapter. The second cDNA synthesis was set up by first incubating the samples at 65℃ for 5 min with 1.7 μM primer (5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGA-3′) and 1 μL dNTPs. Then, the samples were placed on ice and 8 μL of RT reaction mixture was added to each sample. The samples were incubated at 50℃ for 1 h. For second strand cDNA synthesis, in vitro transcription, adaptor ligation, and second cDNA synthesis, purification, and elution were performed after each experiment. Purification was performed using the Agencourt RNAClean XP beads following the manufacturer’s instructions, and elution was performed in 10 mL Milli-Q DNase/RNase-free water. qPCR quantification was performed in 10 μL final volume. The qPCR reaction mixture contained 2 μL of purified sample, 1× qPCR Mix, 0.5 μM PCR InPE1.0 primer (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′), 0.01 μM PCR InPE2.0 primer (5′-GTGACTGGAGTTCA GACGTGTGCTCTTCCGATCT-3′), 0.5 μM PCR Index primer (5′-CAAGCAGA AGACGGCATACGAGATXXXXXXGTGACTGGAGTTC-3′), and 13 EVA green. The conditions were as follows: 98℃ for 3 min and 25 cycles of 98℃ for 20 s, 60℃ for 30 s, and 72℃ for 30 s. qPCR quantification was performed in a final volume of 25 μL with the above conditions plus a final extension at 72℃ for 5 min and the number of cycles identified by qPCR. The final libraries were purified by an automated MBS robot and eluted in 20 mL of Elution Buffer.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
C11CL
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| Data processing |
Thirty-one bases from read 1 were used to determine the spatial barcode and the unique molecular identifier, and 121 bases from read 2 were used to cover the genetic region.
Dimensionality reduction analysis and clustering of the quantitative results of ST were performed using t-distributed Stochastic Neighbor Embedding (t-SNE).
Differential expression analysis was carried out according to the pre-labeled anatomical areas on the brains.
The GO annotation enrichment analyses were performed using the Gene Ontology resource (http://geneontology.org).
Assembly: mm10
Supplementary files format and content: Excel files showing gene expression
Supplementary files format and content: PNG files showing clustering results
Library strategy: Spatial Transcriptomics
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| Submission date |
Oct 05, 2022 |
| Last update date |
Oct 12, 2023 |
| Contact name |
Chaoli Huang |
| E-mail(s) |
chunyang@njmu.edu.cn
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| Phone |
+8613655195884
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| Organization name |
he First Affiliated Hospital of Nanjing Medical University
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| Street address |
300 Guangzhou Road
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| City |
Nanjing |
| State/province |
Jiangsu |
| ZIP/Postal code |
210003 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE214861 |
Myelin-Associated Oligodendrocytic Basic Protein-Dependent Myelin Repair Confers the Long-lasting Antidepressant Effect of Ketamine |
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| Relations |
| BioSample |
SAMN31165902 |
| SRA |
SRX17804765 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6616887_C11CL-celltype.xls.gz |
118.0 Kb |
(ftp)(http) |
XLS |
| GSM6616887_C11CL.png.gz |
510.6 Kb |
(ftp)(http) |
PNG |
| GSM6616887_C11CL_spatial_transcriptomics_celltyping_plot.pdf |
6.1 Mb |
(ftp)(http) |
PDF |
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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