DOI: 10.5061/dryad.v15dv428j
title: Transcriptomic profiles of ligand-stimulated IRF-knockout THP1 cells

description: Interferon regulatory factors (IRFs) are essential for transcription of
 interferons (IFNs), interferon-stimulated genes (ISGs), and
 pro-inflammatory cytokines. We profile the transcriptome of human monocyte
 THP1 cells challenged with cGAMP, LPS, or IFNB1 protein as a function of
 knockout (KO) or overexpression (OE) of IRFs or KO of IFNAR2. We define
 distinct gene expression groups, reflecting the transcription factors
 responsible for their induction, including subgroups activated by more
 than one pathway or feed-forward regulation. We compare IRF3- and
 IRF7-induced gene signatures and note the strong direct induction of a
 subset of antiviral-acting ISGs by IRF3 or IRF7. LPS treatment induces
 NF-kB responses in monocyte and macrophage state cells; however, IFNs and
 ISGs are only co-induced in the macrophage state, requiring IRF3. IRF1,
 IRF2, IRF5, and IRF8 are largely dispensable for IFN-stimulated or
 innate-immune-mediated gene induction. This study provides a valuable
 resource for dissecting complex inflammatory gene signatures and their
 underlying transcription factors, thereby anticipating the effects of
 selectively drugging the underlying pathways.
description: # Transcriptomic profiles of ligand-stimulated IRF-knockout THP1 cells
 Dataset DOI: [10.5061/dryad.v15dv428j](10.5061/dryad.v15dv428j) ##
 Description of the data and file structure We have submitted Supplementary
 Data 1 (Supplementary_Data1.csv), Supplementary Data 2
 (Supplementary_Data2.csv), Supplementary Data 3 (Supplementary_Data3.csv),
 Supplementary Data 4 (Supplementary_Data4.csv), Supplementary Data 5
 (Supplementary_Data5.csv), and Supplementary Data 6
 (Supplementary_Data6.xlsx) **Descriptions and variables** **File:
 Supplementary_Data1.csv** **Description:** Table showing expression values
 (TPM) for various cell lines from this study and published datasets used
 for abundance heatmap generation, shown in Supplementary Figure 1A. #####
 Variables * Gene_Name: Gene symbol * Gene_Category: Gene category (mRNA or
 lncRNA) * InnateGene_Category: Genes assigned to categories within innate
 immune pathways. Genes not fitting into any category are labeled as
 "None". * pDC_untr_SRR3229088_GSE79272: Expression profile (TPM)
 of untreated human Plasmacytoid dendritic cells from SRR3229088_GSE79272.
 * moTHP1_R2_ThisStudy: Expression profile (TPM) of untreated human THP1
 monocytes from this study. * moTHP1_R1_GSM3161742_GSE114966: Expression
 profile (TPM) of untreated human THP1 monocytes from GSM3161742_GSE114966.
 * macTHP1_noLPS_R1_ThisStudy: Expression profile (TPM) of untreated human
 THP1 macrophages from this study. * moTHP1_IFNB1_R2_ThisStudy: Expression
 profile (TPM) of 2 h IFNB1-protein treated human THP1 monocytes from this
 study. * A549_SRR12243954_GSE154596: Expression profile (TPM) of untreated
 human A549 cells from SRR12243954_GSE154596. *
 FB_GSM1712278_GSE69906: Expression profile (TPM) of untreated human
 fibroblast cells from GSM1712278_GSE69906. *
 Hela_SRR9336621_GSE133139: Expression profile (TPM) of untreated human
 Hela cells from SRR9336621_GSE133139. **File: Supplementary_Data2.csv**
 **Description:** Metadata table for the 117 RNA-seq samples described in
 this study. The table includes descriptions for 114 RNA-seq samples used
 for pairwise analysis and clustering analysis in Supplementary Figure 4, 1
 wildtype sample treated with IFNB1 protein for 24 h, and 2 wildtype
 samples treated with the 1x and 100x IFNB1 protein for 2 h, used in
 Supplementary Figure 9A. ##### Variables * Sample_No: Sample number *
 Sample_Name: Sample name * Clustering_inclusion_SuppFig4: Column
 indicating whether the samples were considered for unsupervised clustering
 in Supplementary figure 4 (yes/no) * KO_cells: Column indicating whether
 the samples are wild-type or knockout cell lines *
 Lentiviral_overexpression: Column specifying if the sample overexposes a
 lentivirally transduced gene. Samples without lentiviral transduction are
 labeled as "None".  * PMA_treatment: Column specifying if the
 samples are treated with PMA for differentiation into macrophages.
 "None" indicates no PMA treatment. * Treatment: Column showing
 ligand treatment for each sample (IFNB1, Dig, digCGAMP, LPS).
 "Mock" indicates no ligand treatment. * Replicate: Sample
 replicate * Sequencing_Batch: Sequencing batch * Cell_type: Cell type
 determined based on PMA treatment: mo-monocytes; mac-macrophages *
 RIN_Number: RIN number * Total_read_RD: Total reads for each sample in
 millions * Aligned_RD: Total aligned reads (%) * Aligned_mRNA: Total
 aligned to mRNA (%) * Aligned_rRNA: Total aligned to rRNA (%) *
 Aligned_bact16S: Total aligned to 16S bacterial rRNA (%) *
 RNAseq_filename_R1: Read 1 RNAseq file name for the sample *
 RNAseq_filename_R2: Read 2 RNAseq file name for the sample **File:
 Supplementary_Data3.csv** **Description:** Table showing expression values
 (TPM) for all the samples generated, analyzed, and included in this study,
 expression values (TPM) for public datasets analyzed in our platform and
 described in this study, and DGE analysis results. ##### Variables *
 Gene_Name: Gene symbol * Gene_Category: Gene category (mRNA or lncRNA) *
 Supplementary_Fig4_ClusterGene: Column specifying genes included for
 clustering analysis in Supplementary Figure 4. "Yes' indicates
 included genes and "No" indicates excluded genes. * Gene_Group:
 Group assignment for genes as indicated in Figure 2. Genes without group
 assignment are labeled as "None".  * Gene_Group_subset: Gene
 subgroup assignment. "None" indicates no subgroup assignment. *
 lateISG_IFNB1_24h_log2FC_2_TPM20: late ISGs induced after 24 h IFNB1
 protein treatment (log~2~FC &gt; 2, TPM&gt; 20). * moWT__R1
 to macIRF37DKOlenIRF7_LPS_R1: Expression values (TPM) for all samples.
 Column headers correspond to the sample metadata described in
 Supplementary_Data2.csv.  * DEseq2_log2FC_IFNB1_THP1: Log~2~FC for IFNB1
 protein-treated group vs mock group based on DEseq2 analysis for monocytes
 in this study. * DEseq2_pAdj_IFNB1_THP1: pAdj value for IFNB1
 protein-treated group vs mock group based on DEseq2 analysis for monocytes
 in this study. NA indicates "Not Available". *
 DEseq2_log2FC_Dig: Log~2~FC for digitonin-treated group vs mock group
 based on DEseq2 analysis for monocytes in this study. * DEseq2_pAdj_Dig:
 pAdj value for digitonin-treated group vs mock group based on DEseq2
 analysis for monocytes in this study. NA indicates "Not
 Available". * DEseq2_log2FC_cGAMP: Log~2~FC for
 digitonin/cGAMP-treated group vs digitonin-treated group based on DEseq2
 analysis for monocytes in this study. * DEseq2_pAdj_cGAMP: pAdj value for
 digitonin/cGAMP-treated group vs digitonin-treated group based on DEseq2
 analysis for monocytes in this study. NA indicates "Not
 Available". * DEseq2_log2FC_LPS: Log~2~FC for LPS-treated group vs
 mock group based on DEseq2 analysis for monocytes in this study. *
 DEseq2_pAdj_LPS: pAdj value for LPS-treated group vs mock group based on
 DEseq2 analysis for monocytes in this study. NA indicates "Not
 Available". * DEseq2_log2FC_pma: Log~2~FC for PMA-treated group vs
 mock group based on DEseq2 analysis in this study. * DEseq2_pAdj_pma: pAdj
 value for PMA-treated group vs mock group based on DEseq2 analysis in this
 study. NA indicates "Not Available". * DEseq2_log2FC_pmaLPS:
 Log~2~FC for LPS-treated group vs mock group based on DEseq2 analysis for
 macrophages in this study. * DEseq2_pAdj_pmaLPS: pAdj value for
 LPS-treated group vs mock group based on DEseq2 analysis for macrophages
 in this study. NA indicates "Not Available". * Mock_Avg_TPM:
 Average TPM in Mock group. * IFNB1_Avg_TPM: Average TPM in IFNB1
 protein-treated group. * Dig_Avg_TPM: Average TPM in digitonin-treated
 group. * cGAMP_Avg_TPM: Average TPM in digitonin/cGAMP-treated group. *
 LPS_Avg_TPM: Average TPM in the LPS-treated group. * pma_Avg_TPM: Average
 TPM in PMA-treated group. * pmaLPS_Avg_TPM: Average TPM in LPS-treated
 macrophage group. * ISG_group_log2FC_1_pAdj_05_avgTPM20: Genes unregulated
 by IFNB1 treatment after 2 h ("Up") based on DEseq2 analysis or
 unregulated after 24 h ("lateISG") based on pairwise analysis in
 monocytes. Uninduced genes are labeled as "None". *
 Dig_group_log2FC_1_pAdj_05_avgTPM20: Genes up- or down-regulated by
 digitonin based on DEseq2 analysis in monocytes. Uninduced genes are
 labeled as "None". * cGAMP_group_log2FC_1_pAdj_05_avgTPM20:
 Genes up- or down-regulated by cGAMP based on DEseq2 analysis in
 monocytes. Uninduced genes are labeled as "None". *
 LPS_group_log2FC_1_pAdj_05_avgTPM20: Genes up- or down-regulated by LPS
 based on DEseq2 analysis in monocytes. Uninduced genes are labeled as
 "None". * PMA_group_log2FC_1_pAdj_05_avgTPM20: Genes up- or
 down-regulated by PMA based on DEseq2 analysis. Uninduced genes are
 labeled as "None". * pmaLPS_group_log2FC_1_pAdj_05_avgTPM20:
 Genes up- or down-regulated by LPS in macrophages based on DEseq2
 analysis. Uninduced genes are labeled as "None". *
 log2FCIRF3KO_cGAMP_PC1: Log~2~FC for digitonin/cGAMP-treated IRF3 KO cells
 vs digitonin-only wild-type cells (pseudo count 1) *
 log2FCIRF9KO_cGAMP_PC1: Log~2~FC for digitonin/cGAMP-treated IRF9 KO cells
 vs digitonin-only wild-type cells (pseudo count 1) *
 log2FCIFNAR2KO_cGAMP_PC1: Log~2~FC for digitonin/cGAMP-treated IFNAR2 KO
 cells vs digitonin-only wild-type cells (pseudo count 1) *
 log2FCIRF37DKO_cGAMP_PC1: Log~2~FC for digitonin/cGAMP-treated IRF3/7 DKO
 cells vs digitonin-only wild-type cells (pseudo count 1) *
 log2FCIRF3KO_GFP_cGAMP_PC1: Log~2~FC for digitonin/cGAMP-treated IRF3 KO
 cells overexpressing GFP vs digitonin-only wild-type cells (pseudo count
 1) * log2FCIRF3KO_IRF3_cGAMP_PC1: Log~2~FC for digitonin/cGAMP-treated
 IRF3 KO cells overexpressing IRF3 vs digitonin-only wild-type cells
 (pseudo count 1) * log2FCIRF3KO_IRF7_cGAMP_PC1: Log~2~FC for
 digitonin/cGAMP-treated IRF3 KO cells overexpressing IRF7 vs
 digitonin-only wild-type cells (pseudo count 1) *
 log2FCIRF37DKO_GFP_cGAMP_PC1: Log~2~FC for digitonin/cGAMP-treated IRF3/7
 KO cells overexpressing GFP vs digitonin-only wild-type cells (pseudo
 count 1) * log2FCIRF37DKO_IRF3_cGAMP_PC1: Log~2~FC for
 digitonin/cGAMP-treated IRF3/7 KO cells overexpressing IRF3 vs
 digitonin-only wild-type cells (pseudo count 1) *
 log2FCIRF37DKO_IRF7_cGAMP_PC1: Log~2~FC for digitonin/cGAMP-treated IRF3/7
 KO cells overexpressing IRF7 vs digitonin-only wild-type cells (pseudo
 count 1) * max_log2FC_IRF3OE: Maximum log~2~FC value in IRF3
 overexpressing cells treated with Digitonin/cGAMP. * max_log2FC_IRF7OE:
 Maximum log~2~FC value in IRF7 overexpressing cells treated with
 Digitonin/cGAMP. * AvgMock_16h_Ascano_GSE157052: Average expression values
 (TPM) for mock samples in public dataset GSE157052. *
 AvgcGAMP_16h_Ascano_GSE157052: Average expression values (TPM) for
 cGAMP-treated samples in public dataset GSE157052. *
 GSM5981458_VSVmock_24h: Expression values (TPM) for mock sample in public
 dataset GSM5981458. * GSM5981459_VSV_24h: Expression values (TPM) for
 VSV-treated sample in public dataset GSM5981458. *
 avgTPM_Mono_GSE130011: Average expression values (TPM) for monocyte
 samples in public dataset GSE130011. * avgTPM_M0mac_GSE130011: Average
 expression values (TPM) for M0 macrophage samples in public
 dataset GSE130011. * avgTPM_M1mac_GSE130011: Average expression values
 (TPM) for M1 macrophage samples in public dataset GSE130011. *
 avgTPM_M2mac_GSE130011: Average expression values (TPM) for M2 macrophage
 samples in public dataset GSE130011. * minCount_viremiaISG_GSE176261:
 Minimum counts across the samples for viremia samples in public
 dataset GSE176261. Genes absent in the public dataset are labeled as NR
 (no reads). * maxCount_viremiaISG_GSE176261: Maximum counts across the
 samples for viremia samples in public dataset GSE176261. Genes absent in
 the public dataset are labeled as NR (no reads). *
 avgCount_viremiaISG_GSE176261: Average counts across the samples for
 viremia samples in public dataset GSE176261. Genes absent in the public
 dataset are labeled as NR (no reads). *
 log2FC_viremiaISG_GSE176261: Log~2~FC for viremia samples versus mock
 samples based on DEseq2 analysis in public dataset GSE176261. Genes absent
 in the public dataset are labeled as NR (no reads). NA indicates "Not
 Available". * pAdj_viremiaISG_GSE176261: pAdj values for viremia
 samples versus mock samples based on DEseq2 analysis in public
 dataset GSE176261. Genes absent in the public dataset are labeled as NR
 (no reads). NA indicates "Not Available". *
 viremiaISG_log2FC_1_pAdj_05_avgCount100: Genes identified as ISGs in
 viremic samples are labeled as "yes" (log~2~FC &gt;1, pAdj &lt;
 0.05, and average count &gt;100). * preIFN_moWT_Dig: Expression values
 (TPM) for 24 IFNB1 protein pretreated samples followed by 2 h digitonin
 treatment in wild-type monocytes. * preIFN_moIRF3KO_Dig: Expression values
 (TPM) for 24 IFNB1 protein pretreated sample followed by 2 h digitonin
 treatment in IRF3 KO monocytes. * preIFN_moIRF7KO_Dig: Expression values
 (TPM) for 24 IFNB1 protein pretreated sample followed by 2 h digitonin
 treatment in IRF7 KO monocytes. * preIFN_moIRF37DKO_Dig: Expression values
 (TPM) for 24 IFNB1 protein pretreated sample followed by 2 h digitonin
 treatment in IRF3/7 DKO monocytes. * preIFN_moWT_Dig_cGAMP: Expression
 values (TPM) for 24 IFNB1 protein pretreated sample followed by 2 h
 digitonin/cGAMP treatment in wild-type monocytes. *
 preIFN_moIRF3KO_Dig_cGAMP: Expression values (TPM) for 24 IFNB1 protein
 pretreated sample followed by 2 h digitonin/cGAMP treatment in IRF3 KO
 monocytes. * preIFN_moIRF7KO_Dig_cGAMP: Expression values (TPM) for 24
 IFNB1 protein pretreated sample followed by 2 h digitonin/cGAMP treatment
 in IRF7 KO monocytes. * preIFN_moIRF37DKO_Dig_cGAMP: Expression values
 (TPM) for 24 IFNB1 protein pretreated sample followed by 2 h
 digitonin/cGAMP treatment in IRF3/7 DKO monocytes. **File:
 Supplementary_Data4.csv** **Description:** Table showing ISGs affected by
 the absence of IRF9. ##### Variables * Gene_Name: Gene symbol * Category:
 Gene category (mRNA or lncRNA) * ISG_THP1: Genes identified as ISGs in
 THP1; non-ISGs are labeled as "None". * ISG_Hela: Genes
 identified as ISGs in Hela cells; non-ISGs are labeled as "None"
 * THP1_HeLa_Overlap_ISG: ISGs overlapping between THP1 and Hela cells;
 others are labeled as "None" * IRF9independentISG_THP1:
 IRF9-independent ISGs identified in THP1 cells; others are labeled as
 "None" * moWT__R1_THP1: TPM in untreated THP1 cells (replicate
 1) * moWT__R2_THP1: TPM in untreated THP1 cells (replicate 2) *
 moWT__R3_THP1: TPM in untreated THP1 cells (replicate 3) *
 moWT__R4_THP1: TPM in untreated THP1 cells (replicate 4) *
 moIRF9KO__R1_THP1: TPM in IRF9 KO THP1 cells (replicate 1) *
 moIRF9KO__R2_THP1: TPM in IRF9 KO THP1 cells (replicate 2) *
 moIRF9KO__R3_THP1: TPM in IRF9 KO THP1 cells (replicate 3) *
 moIFNAR2KO__R1_THP1: TPM in IFNAR2 KO THP1 cells (replicate 1) *
 moIFNAR2KO__R2_THP1: TPM in IFNAR2 KO THP1 cells (replicate ) *
 moWT_IFNB1_R1_THP1: TPM in 2 h IFNB1 protein-treated THP1 cells (replicate
 1) * moWT_IFNB1_R2_THP1: TPM in 2 h IFNB1 protein-treated THP1 cells
 (replicate 2) * moIRF9KO_IFNB1_R1_THP1: TPM in 2 h IFNB1 protein-treated
 IRF9 KO THP1 cells (replicate 1) * moIRF9KO_IFNB1_R2_THP1: TPM in 2 h
 IFNB1 protein-treated IRF9 KO THP1 cells (replicate 2) *
 moIFNAR2KO_IFNB1_R1_THP1: TPM in 2 h IFNB1 protein-treated IFNAR2 KO THP1
 cells (replicate 1) * FC_IRF9KO_IFNB1_vs_WT_IFNB1_THP1: Fold change for
 IRF9KO_IFNB1 vs wild-type THP1 (pseudo count 1) *
 FC_IFNAR2KO_IFNB1_vs_WT_IFNB1_THP1: Fold change for IFNAR2KO_IFNB1 vs
 wild-type THP1 (pseudo count 1) * IFNB1_Avg_TPM_THP1: Average TPM in
 IFNB1-treated THP1 cells * DEseq2_log2FC_IFNB1_THP1: Log~2~FC for IFNB1
 protein vs mock based on DEseq2 analysis * DEseq2_pAdj_IFNB1_THP1: pAdj
 value for IFNB1 protein vs mock based on DEseq2 analysis * WT1_NT_HeLa:
 RPM for untreated Hela cells, replicate 1 (GSE118551); genes absent in the
 public processed dataset are labeled as NR (no reads). * WT2_NT_HeLa: RPM
 for untreated Hela cells, replicate 2 (GSE118551); genes absent in the
 public processed dataset are labeled as NR (no reads) * WT1_T_HeLa: RPM
 for IFNB1 protein treated Hela cells, replicate 1 (GSE118551); genes
 absent in the public processed dataset are labeled as NR (no reads) *
 WT2_T_HeLa: RPM for IFNB1 protein treated Hela cells, replicate 2
 (GSE118551); genes absent in the public processed dataset are labeled as
 NR (no reads) * STAT1_KO_NT_HeLa: RPM for untreated STAT1 KO Hela cells
 (GSE118551); genes absent in the public processed dataset are labeled as
 NR (no reads) * STAT1_KO_T_HeLa: RPM for IFNB1 protein treated STAT1 KO
 Hela cells (GSE118551); genes absent in the public processed dataset are
 labeled as NR (no reads) * STAT2_KO_NT_HeLa: RPM for untreated STAT2 KO
 Hela cells (GSE118551); genes absent in the public processed dataset are
 labeled as NR (no reads) * STAT2_KO_T_HeLa: RPM for IFNB1 protein treated
 STAT2 KO Hela cells (GSE118551); genes absent in the public processed
 dataset are labeled as NR (no reads) * DEseq2_log2FC_HeLa: Log~2~FC for
 IFNB1 protein vs mock in wild-type Hela cells based on DEseq2 analysis;
 Genes with no log~2~FC value as determined by DEseq2 are labeled as NA
 (not available). * DEseq2_pAdj_HeLa: pAdj value for IFNB1 protein vs mock
 in wild-type Hela cells based on DEseq2 analysis. Genes with no pAdj value
 as determined by DEseq2 are labeled as NA (not available). *
 avgRPM_WT_IFNB1_HeLa: Average RPM value in IFNB1 protein-treated Hela
 cells * FC_STAT1_IFNB1_Hela_vs_WT_IFNB1_HeLa: Fold change for
 STAT1KO_IFNB1 vs wild-type in Hela cells (pseudo count 1) *
 FC_STAT2_IFNB1_Hela_vs_WT_IFNB1_HeLa: Fold change for STAT2KO_IFNB1 vs
 wild-type in Hela cells (pseudo count 1) **File: Supplementary_Data5.csv**
 **Description:** Table showing gene sets used for cumulative distribution
 function analysis in Supplementary Figure 11. ##### Variables * Gene_Name:
 Gene symbol * Gene_Group: Genes assigned to the groups as shown in Figure
 2. "None" indicates no gene group assignment. * Ctrl_genes:
 Control genes used for the analysis are marked with "X"; all
 others are labeled "None". * Stress_genes: Digitoxin-induced
 stress genes used for the analysis are marked with "X"; all
 others are labeled "None". * NFkB_genes: LPS-induced NF-kB
 targeted genes used for the analysis are marked with "X"; all
 others are labeled "None". * IFNAR2_genes: IFNB1-induced
 IFNAR2-dependent genes used for the analysis are marked with
 "X"; all others are labeled "None". * IRF3_genes:
 cGAMP-induced IRF3-dependent genes used for the analysis are marked with
 "X"; all others are labeled "None". * IIRG: IFNB1
 protein and cGAMP-induced IIRGs used for the analysis are marked with
 "X"; all others are labeled "None". **File:
 Supplementary_Data6.xlsx** **Description:** Numerical source data for
 RT-qPCR plots corresponding to Supplementary Figures 1 and 7. There are
 nine sheets in this file. Each sheet includes values corresponding to each
 Supplementary Figure panel as described below. ##### Sheet name:
 Supplementary_Figure_1D **Description:** C~t~, △C~t~, △△C~t~, and fold
 change values for RT-qPCR samples in Supplementary Figure 1D. ##### Sheet
 name: Supplementary_Figure_1E **Description:** C~t~, △C~t~, △△C~t~, and
 fold change values for RT-qPCR samples in Supplementary Figure 1E. #####
 Sheet name: Supplementary_Figure_1F **Description:** C~t~, △C~t~, △△C~t~,
 and fold change values for RT-qPCR samples in Supplementary Figure 1F.
 ##### Sheet name: Supplementary_Figure_1H **Description:** C~t~, △C~t~,
 △△C~t~, and fold change values for RT-qPCR samples in Supplementary Figure
 1H. ##### Sheet name: Supplementary_Figure_7A_left **Description:** C~t~,
 △C~t~, △△C~t~, and fold change values for RT-qPCR samples in Supplementary
 Figure 7A (left panel). ##### Sheet name: Supplementary_Figure_7A_middle
 **Description:** C~t~, △C~t~, △△C~t~, and fold change values for RT-qPCR
 samples in Supplementary Figure 7A (middle panel). ##### Sheet name:
 Supplementary_Figure_7B_left **Description:** C~t~, △C~t~, △△C~t~, and
 fold change values for RT-qPCR samples in Supplementary Figure 7B (left
 panel). ##### Sheet name: Supplementary_Figure_7B_middle **Description:**
 C~t~, △C~t~, △△C~t~, and fold change values for RT-qPCR samples in
 Supplementary Figure 7B (middle panel). ##### Sheet name:
 Supplementary_Figure_7A_7B_right **Description:** C~t~, △C~t~, △△C~t~, and
 fold change values for RT-qPCR samples in Supplementary Figures 7A (right
 panel) and 7B (right panel). Values shown in red font color represent
 anomalous C~t~ measurement resulting from sample evaporation at the plate
 edges and were therefore excluded from subsequent fold-change calculation.