Name: GSM8215238
Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: The fixed frozen sample slides were baked for 2 hours at 60°C to ensure lung tissue adhered to slides. Following baking, we performed target retrieval for 20 minutes following all recommended settings (MAN-10115-04). RNA targets were exposed using recommended concentration and duration of proteinase K (1ug/ml for 15 min). In situ probe hybridization took place overnight (18 hours) using standard hybridization solution with no custom spike-in (v1.0) Mouse NGS Whole Transcriptome Atlas RNA - lot # MWTA12002). The next day, off target probes were removed using stringent washes as recommended. Finally, morphology markers (SYTO13, B220 – PE, CD3 – CF594, and CD11b eF660) were added. Following antibody staining, slides and collection plate were loaded into GeoMx DSP instrument as recommended (MAN-10152-01). Slides were identified and records created for each. Scan parameters were set for each channel: FITC/525 was utilized for SYTO13 nuclear staining, with exposure time of 50ms. Cy3/568nm was used for Alexa 532 to detect B220. Texas Red/615nm was used for Alexa 594 to detect CD3. Cy5/666nm was used for Cy5 to detect CD11b. All non-nuclear exposures were set for 200ms. Configuration files were obtained from the nanostring website. Syto 13 was used for focus. Slides were then scanned. Multiple ROIs were obtained per lesion, including necrotic core (when applicable), inner lesion, outer lesion border, and full thickness (encompassing inner and outer areas), as assessed by nuclear density and autofluorescence pattern. More fine-grained region determination was not possible due to poor performance of antibody staining. ROIs were then collected. Following collection, GeoMx samples were removed from the machine and allowed to air dry overnight. The following day samples were placed in an open top thermal cycler at 65C for 10 minutes. Next, 10ul of nuclease free water were added to all samples well and pipetted up and down 5 times. PCR was run according to standard GeoMx protocols available in their quick start guide (MAN-10133-03). Pooling and cleanup were also run according to GeoMx protocols, with no deviations. The pooled library was assessed via Bioanalyzer and demonstrated a clean trace. Samples were loaded on the Illumina NextSeq platform at 1.6pM and sequenced twice using the recommended paired end 2 x 27 read acquisition. Sequenced library included 5% PhiX. Fastq files were assessed by QC metrics prior to further analysis.