GSM8285921: P. berghei Infected murine liver, 24h post infection, snRNAseq; Plasmodium berghei ANKA; Mus musculus; RNA-Seq
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