Dneasy blood and tissue kit

Spiked samples were created by combining DNA from multiple human cell lines. Whole genomic DNA derived from B-lymphocytes was purchased from Coriell Institute corresponding to samples NA12878 and NA19129. Additionally, whole genomic DNA was extracted from cultured tumor cells lines A549, NCI-H1975, and MDA-MB-231 (ATCC) using the Qiagen DNeasy Blood and Tissue Kit (catalog number 69504) followed by qPCR quantification using the ThermoFisher RNase P assay (catalog number 4316831). NA19129 was used as the background sample into which the other four samples were spiked. Two different spike levels were created based on DNA concentrations. A projected 1% spike sample was created such that 96% of the DNA mass corresponded to NA19129 and 1% corresponded to each of the four spiked cell lines (S1 Fig). This sample was then diluted 3:1 with NA19129 such that a projected 99% of the DNA corresponded to NA19129 and 0.25% corresponded to each of the four spiked cell lines. A control sample of pure NA19129 was created in order to quantize the error background for ERASE-Seq variant calling. Samples of pure NA12878, MDA-MB-231, NCI-H1975, and A549 were created to determine true variants present in the cell lines.

For genotyping, we first determined whether the tag was inserted into the target locus using PCR. Clones were expanded by splitting cells from individual colonies in 96-well plates into 48-well plates, and subsequently into 24-well plates. The target locus was amplified directly from the cells using the Phire Plant Direct PCR mastermix (ThermoFisher Scientific) in three junction PCR reactions as per manufacturer's protocols. The primers used for these reactions are listed in electronic supplementary material, table S3. The three PCR reactions were designed to amplify the wild-type (WT) locus as well as the left and right junctions of the integration sites. Touchdown PCR was used to improve the quality of the PCR products. Six to twelve positive clones were then expanded into 10 cm dishes for further genotyping. Genomic DNA was extracted using the Qiagen DNeasy Blood and tissue kit, then used to amplify the target locus with Phusion polymerase (ThermoFisher Scientific) by touchdown PCR. The genotype was verified by extracting PCR products and sequencing the tagged and untagged alleles. When possible, homozygous clones carrying only tagged alleles were selected. Alternatively, heterozygous clones carrying a tagged allele and a wild-type allele were selected.

TCR sequencing was carried out by Adaptive Biotechnologies according to standard company protocols. Experimentally, mice from each group were sacrificed and blood draw was obtained through cardiac puncture. Isolation of genomic DNA was performed in the laboratory using a Quiagen DNEasy Blood and Tissue Kit and gDNA quality and concentration was confirmed using a Nano‐Drop fluorimeter (Thermo). Isolated gDNA was then sent to Adaptive headquarters where proprietary primer sets were utilized to isolate and amplify the TCRB gene locus within in mouse genome. Sequencing of the resulting cDNA libraries was performed at Adaptive's facility and PCR duplication bias was eliminated using established company quality controls. Both raw and quantified data were transferred back to the laboratory using Adaptive's data analysis cloud. Raw data were analyzed using MiXCR (Bolotin et al., 2015 (link)) while quantified data were mined and plotting using R (version 4.1).

Nematode isolates of G. pallida, G. rostochiensis, G. tabacum, Heterodera sp. and different mixtures of G. pallida/G. rostochiensis (Table 1) were obtained at the Nematology lab of INIAV (NemaINIAV, Oeiras, Portugal).
The extraction of total DNA was always conducted using the Qiagen DNeasy Blood and Tissue kit (Life Technologies, Carlsbad, CA, USA), following the manufacturer’s instructions. Genomic DNA was quantified using the thermo-NANODROP 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C until further use. DNA extracts were used directly for the PCR reactions without any additional purification step. Total DNA extraction, purification and conservation was performed as described in Camacho et al. [18 (link)].

DNA was extracted from 1×10⁶ cells (QIAGEN DNeasy Blood and Tissue Kit), quantified (Qubit dsDNA BR Assay Kits, Life Technologies), quality controlled (DNA1000 Kit and BioAnalyzer 2100, Agilent), and bisulfite converted (EZ DNA Methylation Kit, Zymo Research) according to each manufacturer's protocol. Bisulfite-converted DNA was hybridized to Infinium-450 BeadChips (Illumina) and scanned with an iScan (Illumina). Quality control was performed in GenomeStudio. HumanMethylation450 data were normalized using SWAN (Maksimovic et al., 2012 (link)) and differential methylation was identified using Limma (Smyth, 2004 (link)). Hierarchical clustering was performed using Cluster, with Euclidian distance and complete linkage. Functional enrichments were executed in GREAT (McLean et al., 2010 (link)) using default settings. DNA methylation data have been deposited in Gene Expression Omnibus under accession number GSE63592.