Pen membrane slide

Tissue specimens were collected from the purple and green leaves. Samples were wrapped using (OCT, Sakura, USA) compound and subsequently cut samples into 20 μm sections using a cryomicrotome (Leica CM3050S, Germany) at − 20 °C. The tissue sections were then uniformly adhered to PEN membrane slides (Leica). Segmentation of the tissue sections into upper and lower parts using an automated laser microdissection system (Leica, LMD7000) [28 (link)].

Grain samples from H. vulgare cv. Sloop were collected at 11 and 25 DPA, bisected transversely and fixed in ethanol:acetic acid as described previously³¹ . Tissues were embedded in butyl methyl methacrylate (BMM) and polymerised at −20 °C under UV light^{31 ,32 (link)}. Samples were sectioned to 5 µm using a Leica Ultracut microtome, adhered to Leica PEN membrane slides and dissected using a Leica LMD microscope (Leica, Wetzlar, Germany; Adelaide Microscopy, Adelaide; Fig. S2). Approximately 6–10 sections from three grain were collected from the outer grain layers (predominantly pericarp), aleurone, outer-starchy endosperm (incorporating sub-aleurone and some adjoining starchy cells) and inner starchy endosperm (incorporating starchy endosperm cells and the grain cavity) and stored at −80 °C. Total RNA was isolated using the PicoPure kit (ThermoFisher, Australia) and converted to cDNA using Superscript^TM III reverse transcriptase (ThermoFisher, Australia) and oligodT primer with a 2 hour synthesis step at 37 °C. For the 25 DPA samples, RNA was amplified twice using the MessageAmp^TM II kit (ThermoFisher, Australia) before converting to cDNA using Superscript III and random hexamers²⁷ . Multiple control genes were used to normalise samples^{33 (link)} and primer sequences are included in Table S5.

LCM was performed on the PDX as previously described [11 (link)]. Briefly, cresyl violet stained slides were brought to room temperature. Tumour cells from each cresyl violet section were microdissected using the PALM LMPC device (Carl Zeiss MicroImaging, GmbH, Munich, Germany). Tissue was collected in AdhesiveCap tubes (Carl Zeiss MicroImaging, GmbH, Munich, Germany) and stored at -80°C prior to extraction.
LCM of fresh frozen tissue samples from PDAC was performed on a Leica LMD 7000 instrument. Frozen tissue for tumour samples was maintained in vapor-phase liquid nitrogen and embedded in OCT cutting medium and sectioned in a cryotome into 8-μm thick sections. These sections were then mounted on PEN membrane slides (Leica) and lightly stained with hematoxylin to distinguish tumour epithelium from stroma. A pathologist (SF) marked tumour sections and LCM was performed on the same day according to manufacturer’s protocol on the Leica LMD7000 system. Microdissected tumour cells were collected by gravity into the caps of sterile, RNAse-free microcentrifuge tubes. Approximately 150,000–200,000 tumour cells were collected for each DNA extraction and stored at –80°C in Arcturus PicoPure Extraction Buffer.

Target cell populations (i.e. PR-high and PR-low cells) were microdissected using Leica LMD 6 Laser Capture Microdissection system (Leica Microsystems CMS GmbH) equipped by Leica’s DFC7000 T camera (resolution of 1920 × 1440 pixels and pixel size of 4.54 × 4.54 µm). The cells were microdissected from immunocytochemically stained MCF-7 FFPE sections adhered to 2.0 µm PEN-membrane slides (Leica). Single cells were dissected at 63X magnification (HCX PL FLUOTAR 63X lens, numerical aperture 0.7). The laser beam settings were adjusted to power 20, aperture 1, and speed 25. 2000–3000 cells were collected from each target population onto the caps of 0.2 ml PCR tubes and preserved at room temperature until extraction. One sample of 2000 cells was collected from each population for qRT-PCR. Samples for whole transcriptome RNA-seq were microdissected from three biological replicates of MCF-7 FFPE preparations. PR-high and PR-low cells population were microdissected from each MCF-7 FFPE preparation; each population containing 3000 microdissected cells.
Triplicate bulk, unsorted MCF-7 cell line samples were harvested in parallel for bulk RNA-seq analysis to emphasize the difference between the common gene expression averaging in bulk analysis as compared to high-resolution analysis using ICC-RNAseq.

PLP-fixed frozen samples were cut in a cryostat at −22 °C into 5-μm sections and mounted on PEN-Membrane slides (Leica, Wetzlar, Germany). The tissue sections were stained with mouse anti-human PD1 (NAT105 ab52587, Abcam, Cambridge, UK) and anti-human CD20cy (clone L26, Dako, Michigan, MI, USA) antibodies, diluted 1:2000 and 1:1000, respectively, and detected by use of the Envision⁺ Dual Link System-HRP (Dako). The tissue sections were then counterstained with hematoxylin (Mayer's hematoxylin, Muto Pure Chemical, Tokyo, Japan) for 20 s at room temperature. After staining, tissue sections were dehydrated with ethanol and dried at room temperature before laser microdissection (LMD).