GSM8463389: Pancreas, Acini_Distal, 8; Mus musculus; OTHER
Slides were prepared following NanoString GeoMx DSP Manual RNA Assay Slide Preparation. In the scan area of each slide, two 5-μm-thick FFPE tissue sections were placed, for a total of four mice. Slides were incubated with Whole Transcriptome Atlas Probe Mix overnight. For segmentation of the different cell populations, slides were incubated with the following morphology markers: Reg3a/g (B-10) (Santacruz biotechnologies, sc-377038), Amylase (D55H10) XP (CST, 3796), PanCK AlexaFluor 532 (Novus Biologicals, NBP2-33200AF532) and SYTO 13 (Invitrogen, S7575) for nuclear staining. Slides were then scanned with GeoMx Digital Spatial Profiler (Nanostring). Regions of interest (ROIs) were selected based the morphology markers and a total of 95 segments were collected for analysis. After the GeoMx DSP system has completed collection, the DSP collection plate can be removed from the instrument. Each GeoMx DSP aspirate in the plate contains photocleaved DNA oligos comprised of an analyte identifier, a unique molecular identifier (UMI) barcode, and primer binding sites. 95 ROIs were dried down at 65C for 1 hour on a PCR block and resuspended in 10ul of Nuclease-Free water for 10 minutes. PCR was then performed with the matching primers from the primer plate Seq Code C. When PCR is performed on the aspirates, Illumina adapter sequences and unique dual sample indices are added. After the PCR step, 4ul of each library were pooled, size selected and clean-up using AMPure XP beads (Beckman Coulter, #A63881). 4ul of each library was pooled, creating a volume of 380ul. 456ul of Ampure was added to the first step of the size selection. The final elution volume was 48ul. The final library had an average peak size of 153bp. The library pool was quantified by fluorometry using the Qubit dsDNA HS assay and assessed for quality and fragment size using the Agilent Bioanalyser with the DNA HS Kit (#5067-4626). Fragment size and quantity measurements were used to calculate molarity. The pool had a molarity of 29.7nM.