|
| Status |
Public on Jun 03, 2024 |
| Title |
P. berghei Infected murine liver, 24h post infection, snRNAseq |
| Sample type |
SRA |
| |
|
| Source name |
Liver
|
| Organisms |
Plasmodium berghei ANKA; Mus musculus |
| Characteristics |
tissue: Liver
treatment: P. berghei infected
time: 24h post infection
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The livers were collected, and lobes were separated. Each lobe was segmented and frozen in -30°C 2-Methylbutane. Frozen liver tissues were homogenized using the Kimble Dounce grinder in homogenization buffer with RNAse inhibitors. Nuclei were isolated from snap frozen liver tissue with a sucrose gradient. Homogenized tissue was then subjected to density gradient (29% cushion – Optiprep) ultracentrifugation. Nuclei were resuspended and 2 biological replicates of each condition were pooled before nuclei were stained using DAPI. Intact nuclei were FACS-purified from remaining debris and sorted into BSA coated tubes. The sorted nuclei were pelleted by centrifugation and then resuspended in PBS with 0.04% BSA.
Nuclei suspensions were loaded on a GemCode Single-Cell Instrument (10x Genomics) to generate single-cell Gel Bead-in-Emulsions (GEMs). Single-cell RNA-Seq libraries were prepared using GemCode Single-Cell 3ʹGel Bead and Library Kit (10x Genomics, V2 and V3 technology) according to the manufacturer’s instructions. Briefly, GEM-RT was performed in a 96-Deep Well Reaction Module. After RT, GEMs were broken down and the cDNA was cleaned up with DynaBeads MyOne Silane Beads and SPRIselect Reagent Kit. cDNA was amplified with 96-Deep Well Reaction Module. Amplified cDNA product was cleaned up with SPRIselect Reagent Kit prior to enzymatic fragmentation. Indexed sequencing libraries were generated using the reagents in the GemCode Single-Cell 3ʹ Library Kit with the following intermediates: (1) end repair; (2) A-tailing; (3) adapter ligation; (4) post-ligation SPRIselect cleanup and (5) sample index PCR. Pre-fragmentation and post-sample index PCR samples were analyzed using the Agilent 2100 Bioanalyzer. snRNA-seq libraries were pooled in equal ratios and loaded on a S4 lane Illumina NovaSeq 6000, resulting in 2500 - 3000 million read-pairs.
single-nuclei RNA sequencing
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10x Genomics
|
| Data processing |
The demultiplexing, barcode processing, gene counting and aggregation steps were carried out using the Cell Ranger software v3.1.0
Assembly: custom reference genome combining Mus musculus (GRCm38.101) and Plasmodium berghei (PlasmoDB-48_PbergheiANKA)
Supplementary files format and content: Matrix files
|
| |
|
| Submission date |
May 22, 2024 |
| Last update date |
Jun 03, 2024 |
| Contact name |
Miren Urrutia Iturritza |
| E-mail(s) |
miren.urrutiaiturritza@su.se
|
| Organization name |
Stockholm University
|
| Department |
The Department of Molecular Biosciences, The Wenner-Gren Institute (MBW)
|
| Lab |
Ankarklev Lab
|
| Street address |
Svante Arrhenius väg 20C
|
| City |
Stockholm |
| ZIP/Postal code |
114 18 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL34512 |
| Series (1) |
| GSE268112 |
A Spatial Transcriptomics Atlas of the Malaria-infected Liver Indicates a Crucial Role for Lipid Metabolism and Hotspots of Inflammatory Cell Infiltration |
|
| Relations |
| BioSample |
SAMN41492153 |
| SRA |
SRX24651623 |
