 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 15, 2024 |
| Title |
Brain, Uninfected, rep2, GEX |
| Sample type |
SRA |
| |
|
| Source name |
Brain
|
| Organism |
Macaca mulatta |
| Characteristics |
tissue: Brain
cell type: CD11b positive cells
|
| Treatment protocol |
Two rhesus macaques (21T and 34T) were intravenously inoculated with SIVmac251 and infection proceeded for 92 and 49 days, respectively. The other two rhesus macatues (104T and 106T) were uninfected controls
|
| Extracted molecule |
total RNA |
| Extraction protocol |
A necropsy was performed on deeply anesthetized (ketamine plus xylazine) animals, following intracardial perfusion with sterile PBS containing 1 U/ml heparin. Brains were harvested, and approximately half of the brain was taken for microglia/macrophage isolation. Briefly, the brain was minced and homogenized in cold Hank’s Balanced Salt Solution (HBSS). After being centrifuged, the brain tissue was digested at 37° C in HBSS containing 28 U/ml DNase I and 8 U/ml papain for 30 minutes. After digestion, the enzymes were inactivated by adding 2.5% FBS, and the cells were centrifuged and resuspended in cold HBSS. The cell suspension was mixed with 90% Percoll to yield a final concentration of 20% Percoll and centrifuged at 4° C for 15 minutes at 550 x g. The microglia/macrophage pallet at the bottom was resuspended in HBSS and passed through a 40 μm strainer to remove cell clumps and aggregates. Cells were again pelleted by centrifugation and resuspended in RBC lysis buffer for 3 minutes to eliminate contaminating red blood cells. A final wash was performed before the resulting cells were quantified on a hemocytometer and Coulter Counter Z1. The cells were resuspended in 10% DMSO and 90% fetal bovine serum and subjected to slow controlled freezing followed by storage in liquid N2. On the day of the experiment, cryopreserved cell isolates were rapidly thawed in a 37° C water bath. After the recovery, cells were washed and counted by Coulter Counter Z1. Once cell concentration was known, cells were transferred to ice-cold PBS and stained with CD11b and UV-blue live/dead. Cells were washed, resuspended in a flow cytometry staining buffer, and sorted on an Aria2 flow cytometer and then processed for the Multiome technique
Nuclei were isolated from the cells and permeabilized as the 10XGenomics protocol. Nuclei were then quantified on a hemocytometer and concentrated to approximately 2200-2400 nuclei per µL. Based on 10× Genomics parameters targeting 8000 nuclei, the ideal volume of cells was loaded onto the 10× Genomics (Pleasanton, CA, USA) Chromium Next GEM Chip J and placed into the Chromium Controller for nuclei capturing and library preparation. The prepared libraries were sequenced using Illumina (San Diego, CA, USA) Novaseq6000 sequencers.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
The demultiplexing, barcoded processing, and gene counting were made using the Cell Ranger arc software v2.0.2
Assembly: Mmul10, NCBI RefSeq assembly plus SIVmac251 sequence (Gene bank assession number: PP236443)
Supplementary files format and content: FASTQ files contain the sequenced data
Supplementary files format and content: Fragment files contain reference genome chromosome of fragment, ajusted start and end position of fragment on chromosome, cell barcode, and total number of read pairs associated with this fragment
|
| |
|
| Submission date |
Jul 19, 2024 |
| Last update date |
Oct 15, 2024 |
| Contact name |
Xiaoke Xu |
| E-mail(s) |
x.xu@unmc.edu
|
| Organization name |
University of Nebraska Medical Center
|
| Street address |
42nd and Emile
|
| City |
Omaha |
| State/province |
Nebraska |
| ZIP/Postal code |
68198-7400 |
| Country |
USA |
| |
|
| Platform ID |
GPL27943 |
| Series (1) |
| GSE272669 |
The single-cell assessment of transcriptiomic profile and chromain accessibility for the brain myeloid cells in rhesus macaques with SIV-induced encephalitis |
|
| Relations |
| BioSample |
SAMN42652037 |
| SRA |
SRX25389148 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8408912_106T_GEX_filtered_feature_bc_matrix.h5 |
97.6 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
