title: GSM8542373: Low dose infection 12 hours; Mus musculus; RNA-Seq
library_construction_protocol: Peripheral blood was collected from the orbital vein into EDTA anticoagulant tubes at each time point, and the blood samples were gently inverted to ensure thorough mixing with the anticoagulant. Approximately 300-400 µL of blood was collected from each mouse, with samples from 5 mice per time point pooled together in a new anticoagulant tube for subsequent extraction of peripheral blood mononuclear cells.Transfer 4 ml of density gradient centrifugation fluid into a density gradient centrifugation tube and allow it to equilibrate to room temperature. Carefully add 1 ml of blood sample slowly onto the top layer of the centrifugation fluid using a pipette. Then, initiate centrifugation without braking mode at 400 x g for 30 minutes. After centrifugation, gently aspirate the PBMC layer cells into a new 15 ml centrifuge tube and add 9 ml of serum-free RPMI1640 culture medium for washing, followed by centrifugation at 250 x g for 10 minutes. Discard the supernatant, add 1 ml of red blood cell lysis reagent to each tube, and incubate for 5 minutes. Then, add 9 ml of RPMI1640 culture medium, mix well, and centrifuge at 250 x g for 10 minutes. Discard the supernatant, add 2 ml of RPMI1640 to resuspend the pellet, and filter through a sieve to remove cell clumps. Add another 3 ml of RPMI1640 to wash the original tube and filter again. Transfer 5 ml of PBMC suspension to a new 15 ml centrifuge tube and centrifuge at 250 x g for 10 minutes. Discard the supernatant and resuspend the cells in 500 μl of PBS containing 1% BSA to obtain mouse peripheral blood mononuclear cells (PBMCs). The single-cell RNA-Seq libraries were prepared using the Chromium™ Single Cell 3' Solution kit from 10×Genomics. In brief, single-cell suspensions were combined with a mixture containing gel beads encapsulating Barcode information, Unique Molecular Identifier (UMI) information, Poly(dT)VN capture sequences, and primers, and then entered into a microfluidic device where they were encapsulated in oil droplets, forming the Gel Beads-In-Emulsions (GEMs). Subsequently, the gel beads inside the GEMs were dissolved, releasing the 3'-end mRNA sequences captured by Poly(dT)VN, which were reverse transcribed to generate cDNA molecules tagged with Barcodes and UMIs for sequencing. After disrupting the emulsion, a cDNA amplification reaction was performed, followed by purification, quality assessment, and enzymatic fragmentation of the qualified cDNA samples. The 3' ends of the fragmented cDNA were blunt-ended and adenylated. Additionally, P5 and P7 adapter sequences were ligated to the 5' and 3' ends of the fragments, respectively. Subsequently, target-sized fragments were selected and recovered, followed by quality control and quantification of the sequencing libraries. Finally, the prepared libraries were subjected to high-throughput sequencing using the Illumina sequencing platform with paired-end 150-base pair reads.