Thirty-five tumor biopsies were collected prior to and/or within 7 days following SBRT, but prior to pembrolizumab, from 24 patients: 19 pre-SBRT and 16 post-SBRT (11 matched samples from the same tumor) (Table S1 ). RNA was extracted and analyzed using Affymetrix Human Clariom™ D microarrays. Computational gene expression deconvolution methods were used for genome-wide expression analyses following SBRT and correlated with irradiated tumor response. We utilized Ingenuity Pathway Analysis (IPA) to predict the upstream regulators of pathways involved in the gene expression changes. See Supplemental Data for details regarding baseline PD-L1 expression, microarray data pre-processing, and detection of treatment-responsive gene expression changes. A TaqMan RT-PCR assay (ThermoFisher) was used to validate DNASE1 gene expression utilizing an exon-spanning probe (Hs00173736_m1) overlapping the Clariom D transcript cluster TC1600011345.hg. Expression values for DNASE1 were normalized to the geometric mean of β-actin and 18S rRNA.
Human clariom d microarrays
Manufactured by Thermo Fisher Scientific
Sourced in Sweden, Spain
The Human Clariom™ D microarrays are high-density microarray products designed for transcriptome analysis. They provide comprehensive coverage of the human transcriptome, enabling the detection and quantification of coding and non-coding RNA expression.
Automatically generated - may contain errors
Lab products found in correlation
Taqman rt pcr assay, by Thermo Fisher Scientific (1 mentions)
Rlt cell lysis buffer, by Qiagen (1 mentions)
Dulbecco s modified eagle s medium dmem, by Lonza (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Six well plate, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Lonza (1 mentions)
Cyclopiazonic acid cpa, by Merck Group (1 mentions)
Ht29 cell line, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Genematrix universal rna purification kit, by EURx (1 mentions)
Ncm460 cells, by INCELL (1 mentions)
Polyaminered, by Funakoshi (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
4 protocols using human clariom d microarrays
1
Transcriptional Changes Following SBRT
2
Transcriptomic Analysis of GC B Cells
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DAFhi and DAFlo GC B cells (CD19+ CD20+ CD38+ IgD-) were resuspended in RLT cell lysis buffer (Qiagen) after flow cytometric sorting. Total RNA was extracted (Qiagen) and microarray was performed using Affymetrix Human Clariom D microarrays (Bioinformatics and Expression Analysis core facility at Karolinska Institutet, Huddinge, Sweden). Data were analyzed in R. First the data was RMA normalized. Next, limma was used to solve the differential expression regression problems using empirical Bayes. In all cases we regressed out donor effects (~x+donor, where x is e.g. DAFhi vs DAFlo).
3
Cell Culture Optimization for Cancer Research
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The HT29 cell line was obtained from LONZA (Basel, Switzerland). NCM460 cells were from INCELL Corporation (San Antonio, TX, USA). SW480 cells were a kind gift from Prof. Alberto Muñoz (CSIC, Madrid, Spain). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, and fetal bovine serum (FBS) were sourced from Lonza (Basel, Switzerland). L-glutamine was from Gibco (Barcelona, Spain). Trypsin-EDTA was from LONZA (Verbiers, Belgium). Poly-L-Lysin was from Marlenfeld GmbH (Lauda-Könlgshofen, Germany). Six-well plates were from NUNC (Thermo Scientific, Waltham, MA, USA). Dishes 10 cm2 in diameter were from Corning (NY, USA). DFMO was from TOCRIS (Bristol, UK). Fura2/AM and qPCR primers are from Invitrogen (Eugene, OR, USA). Cyclopiazonic acid (CPA) was from Sigma-Aldrich (Steinheim, Alemania). Antibodies against MCU and β actin were from Sigma (Madrid, Spain). The RNA extraction kit was a GeneMATRIX Universal RNA Purification Kit from EURx (Gdansk, Poland). Clariom D human microarrays (Affymetrix) were supplied by CABIMER (Andalucía, Spain). RNA-seq (Illumina) was provided by Sistemas Genómicos S.L (Valencia, Spain). PolyamineRED was from Funakoshi Co., Ltd., Tokyo, Japan). All other reagents were obtained from Sigma and Merck.
4
Profiling miRNA and gene expression
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Total RNA was extracted from cell line material using Trizol in accordance with the manufacturer’s instructions (Life Technologies, Paisley, UK). One µg of total RNA was used for Affymetrix Genechip miRNA v.4.0 microarrays, and 200 ng of DNAse treated total RNA were used for Clariom D human microarrays to measure miRNA and gene expression, respectively. The RNA was labelled and hybridised to microarrays in accordance with the manufacturer’s instructions (Affymetrix, CA, USA).
Resulting raw intensity data (i.e., cel files) were imported and analysed within Transcriptome Analysis Console (TAC) software version 4.0.2 (Affymetrix, CA, USA). Using this software, we identified differentially expressed miRNAs or genes on the basis of >2-fold up- or down-regulation along with Benjamini–Hochberg multiple corrected p values < 0.05. All microarray data was MIAME compliant, and raw data was deposited in the GEO database (GSE183140). For miRNA microarray analysis, probes were filtered for only human mature miRNAs (i.e., hsa-miR*) (* means wild-card i.e., any miR) and, for gene expression analysis coding, genes were classified as probes and filtered using the group variable ‘coding’ or ‘multiple complex’, before removing non-annotated genes that only had a numerical Aceview description.
Resulting raw intensity data (i.e., cel files) were imported and analysed within Transcriptome Analysis Console (TAC) software version 4.0.2 (Affymetrix, CA, USA). Using this software, we identified differentially expressed miRNAs or genes on the basis of >2-fold up- or down-regulation along with Benjamini–Hochberg multiple corrected p values < 0.05. All microarray data was MIAME compliant, and raw data was deposited in the GEO database (GSE183140). For miRNA microarray analysis, probes were filtered for only human mature miRNAs (i.e., hsa-miR*) (* means wild-card i.e., any miR) and, for gene expression analysis coding, genes were classified as probes and filtered using the group variable ‘coding’ or ‘multiple complex’, before removing non-annotated genes that only had a numerical Aceview description.
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