Spatial Transcriptomic Experiment of Triple-Negative Breast Cancer PDX Model PIM001-P model treatment naive sample

Spatial Transcriptomic Experiment of Triple-Negative Breast Cancer PDX Model PIM001-P model treatment naive sample

10X Genomics Visium platform. 

Library Preparation and Sequencing of PIM001P

Tissue sections of 10µm thickness were mounted onto the capture areas of the Visium Spatial Gene Expression slide and stained using hematoxylin and eosin. Tissue sections were permeabilized on a thermocycler for 24 minutes, as determined by the Tissue Optimization step. Poly-adenlyated mRNA is released and captured by surface-bound primers within each capture area. Reverse transcription, template switching, extension, and second strand synthesis are performed on the slide. Full-length, spatially barcoded cDNA transcripts are then denatured from the slide and amplified via PCR prior to library construction. Approximately 110 to 375 ng of amplified cDNA was carried forward into library construction. During library construction, cDNA is enzymatically fragmented to target amplicon size then undergoes end repair, A-tailing, adapter ligation, and then amplified using between 14 and 16 PCR cycles. The resulting libraries were quantitated using the Invitrogen Qubit 2.0 quantitation assay and fragment size assessed with the Agilent Bioanalyzer. A qPCR quantitation was performed on the libraries to determine the concentration of adapter ligated fragments using Applied Biosystems ViiA7 Real-Time PCR System and a KAPA Library Quant Kit (p/n KK4824). All samples were pooled equimolarly and re-quantitated by qPCR, and also re-assessed on the Bioanalyzer.

Sequencing:

150 pM of equimolarly pooled library was loaded onto the NovaSeq 6000 S4 flowcell and sequenced at the recommended 28-10-10-50 read configuration. PhiX Control v3 adapter-ligated library (Illumina p/n FC-110-3001) was spiked-in at 2% by weight to ensure balanced diversity and to monitor clustering and sequencing performance. A minimum of 300 million read pairs per sample was sequenced. FastQ file generation was executed using 10X Genomics’ Space Ranger mkfastq software.