GSM8900556: Lung, vaccinated, rep1; Mus musculus; OTHER
Sample preparation for mouse lung for spatial transcriptomics To prepare for tissue collection, isopentane was chilled in a metal beaker placed on dry ice to ensure rapid tissue freezing. Lung samples were collected from both naïve and vaccinated mice (n=4 per group) based on a published protocol (20). Mice were sacrificed, and a laparotomy was performed to expose the thoracic cavity.  Lungs were perfused via a tracheal needle with an Optimal Cutting Temperature (OCT)/PBS solution (1:1) (20). After inflation, extracted lungs were rinsed in OCT, coated with room-temperature OCT in a pre-cooled cryomold, and placed in chilled isopentane until OCT solidified and turned white (20). Samples were stored at -80°C for subsequent cryo-sectioning and analysis. Tissue sectioning, staining and imaging Tissue samples were cut using a Leica CM3050 S cryostat (Leica Biosystems, Deer Park, IL), and 10 µm thick sections were placed on capture areas of Visium spatial gene expression slides v1 (10X Genomics, Pleasanton, CA), as described previously  (21). Slides were fixed in pre-chilled methanol (-20°C) for 30 minutes and stained with Mayer's hematoxylin for 7 minutes. Sections were washed in nuclease-free water, incubated in bluing buffer (Dako) for 2 minutes, counterstained with eosin for 1 minute, briefly rinsed, and dried on a slide warmer at 37°C for 5 minutes. Images of individual capture areas with stained sections were acquired using a Zeiss Axiovert 200M motorized microscope (Carl Zeiss Microscopy, White Plains, NY) with a 5x Plan Apochromat objective and a Leica DFC290 camera. Image tiles were stitched using Metamorph software (Molecular Devices, LLC, San Jose, CA). The pixel size of the final composite images was 1.275 µm. Permeabilization, reverse transcription, second strand synthesis, and cDNA PCR amplification. The optimal enzymatic permeabilization time for the tissue type was pre-determined using the 10X Genomics Tissue Optimization kit, as described previously (21).  The stained and imaged slides were incubated in the permeabilization enzyme solution for 8 min at 37˚C.  Following the permeabilization wash step, the reverse transcription master mixture was added and incubated for 45 min at 53˚C.  After degradation of the mRNA with KOH, the second strand synthesis was carried out for 15 minutes at 65˚C by adding a second strand mixture containing the second strand enzyme and primer. The second strand cDNA was released by denaturation of the double-stranded cDNA with KOH, neutralized with 1 M Tris-HCl and transferred from the slide section to an 8-tube strip.  After determinating the optimal PCR cycle number by KAPA SYBR FAST qPCR Master Kit, the cDNA was amplified for 15 cycles using the PCR cycling conditions provided in the 10X Genomics Visium Spatial Gene Expression protocol. The cDNA was purified using 0.6X AxyPrep Mag magnetic beads. The quality and concentration were confirmed by the Fragment Analyzer (Agilent) using the High Sensitivity NGS kit. The cDNA library for the Illumina platform was prepared by following the 10X Genomics spatial gene expression library construction protocol, as described previously (21).  Briefly, the double-stranded cDNA was fragmented, the end was repaired, and an A-tail was added in a single step.  The A-tailed fragments were size selected using double-sided AxyPrep Mag magnetic beads with a bead-to-reaction solution ratio of 0.6X for the first step and 0.8X for the second step. The adaptor was ligated and purified with 0.8X AxyPrep Mag magnetic beads. The final Illumina libraries were amplified by PCR, which added the sample dual indexes.  The PCR amplified libraries were purified and size selected using double-sided AxyPrep Mag magnetic beads with a bead-to-reaction solution ratio of 0.6X for the first step and 0.8X for the second step. The individual library was analyzed by Fragment Analyzer (Agilent) to determine the quality and average fragment size and quantified using the Qubit (Invitrogen) DNA assay.  The libraries were pooled in equal molar ratios with a total concentration of 5 nM and sequenced on the Illumina Novaseq 6000.