The plant material was collected from Agastya hills, Trivandrum, Kerala (India). Genomic DNA was isolated from tender leaf tissues using CTAB method followed by 0.5 bead purification twice for both Illumina and Pacbio sequencing (Doyle and Doyle (1987) ). The quality of the DNA sample was assessed using 0.75% agarose gel assay and Nanodrop (Nanodrop Technologies, Wilmington, DE, US), and was quantified using Qubit system (Thermo Fisher Scientific, Waltham, MA).
Qubit system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Canada
The Qubit system is a high-sensitivity fluorometer designed for the quantification of DNA, RNA, and protein samples. It provides accurate and precise measurements of nucleic acid or protein concentrations in small sample volumes. The Qubit system uses fluorescent dyes that specifically bind to the target molecule, enabling accurate quantification even in the presence of contaminants.
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Lab products found in correlation
Tapestation 2200, by Agilent Technologies (7 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Kapa library quantification kit, by Avantor (5 mentions)
7900ht sequence detection system, by Thermo Fisher Scientific (5 mentions)
Experion dna 1k chip, by Bio-Rad (5 mentions)
Nextseq 500 platform, by Illumina (5 mentions)
Miseq system, by Illumina (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Nextseq500 high output kit 300 cycles pe100, by Illumina (4 mentions)
Basespace onsite computing platform, by Illumina (4 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Nanodrop one, by Thermo Fisher Scientific (4 mentions)
Tapestation 4200, by Agilent Technologies (3 mentions)
Ampure xp bead, by Beckman Coulter (3 mentions)
Novaseq 6000, by Illumina (3 mentions)
S2 ultrasonicator, by Covaris (3 mentions)
Bioanalyzer, by Agilent Technologies (3 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Miseq platform, by Illumina (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
88 protocols using qubit system
1
Agastya Hills Genomic DNA Extraction
2
High Molecular Weight DNA Extraction and Sequencing
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For the extraction of high molecular weight DNA, cells were disrupted using the Tissuelyser II (Qiagen) and Lysing Matrix E (MP Biomedicals) and DNA was purified using the NucleoSpin Food kit (Macherey-Nagel). DNA quality was assessed using the Agilent FEMTOpulse system and DNA quantity was measured using the Thermo Scientific Qubit system. PacBio libraries were prepared from unfragmented DNA with the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences) followed by size selection for enrichment of fragments ≥10 kb (Blue Pippin, SAGE Science). After purification with magnetic beads, the library was sequenced on the Sequel with Sequel DNA polymerase and binding kit version 3.0 and sequencing chemistry version 3.0 for 1200 min. Data was collected from two SMRT cells, with a total output of 8.86 Gb (Supplementary Note 1 ).
3
Targeted Gene Sequencing of Organoid Samples
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Organoids were collected in cell recovery solution (Corning, Corning, NY, USA). Genomic DNA was extracted with the QIAamp DNA Mini kit (Qiagen, Valencia, CA, USA), and the DNA quality was analyzed using the Qubit system (Thermo Fisher Scientific). An amount of 200 ng of genomic DNA was used in library construction via the HaloPlex kit (Agilent, Santa Clara, CA, USA), according to the manufacturer's instructions. Target genes were selected based on reported recurrent gene mutations.8 , 9 A list of target genes is given in Table S1 . Sequencing was performed by on the MiSeq system (Illumina, San Diego, CA, USA). Fastq files were mapped and analyzed using SureCall (Agilent). APC variants were culled by removing those registered in the Human Genetic Variation Database (HGVD release version 2.30). Other variants were annotated with ANNOVAR (2015‐03‐22 released).10 The variants were excluded from the following analysis when maximal population frequency is over 1%. We excluded somatic missense SNVs with the following properties; (a) variants were neither deposited on COSMIC (ver82 https://cancer.sanger.ac.uk/cosmic/ ), (b) any variant fraction of the same patients does not exceed 0.25 (subclonal mutations).
4
Quantitative Protein Analysis using Qubit
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Protein quantification was determined using a fluorimeter Qubit System (Thermo
Fisher) using the reagents of the Qubit Protein Assay kit as recommended by the
manufacturer.
Fisher) using the reagents of the Qubit Protein Assay kit as recommended by the
manufacturer.
5
DNA Extraction from FFPE and Frozen Tissues
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DNA preparation from FFPE samples was performed by using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), the GeneRead DNA FFPE Kit (Qiagen), or the Maxwell® RSC FFPE Plus DNA Kit together with the Maxwell® RSC instrument (Promega, Mannheim, Germany). DNA extraction from frozen tissue samples was performed with the PureLink™ Genomic DNA Mini Kit (Life Technologies, Carlsbad, CA, USA) or by ultracentrifugation as described elsewhere [27 (link)]. Peripheral blood leukocyte DNA was extracted either with the PureLink™ Genomic DNA Mini Kit (Life Technologies) or with the Maxwell® RSC Blood DNA Kit. Extracted DNA was quantified by using the Quantus™ Fluorometer (Promega) or the Qubit system (ThermoFisher Scientific, Waltham, MA, USA).
6
Comprehensive mtGenome Assembly Protocol
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The DNA concentration was evaluated using the Qubit system (Thermo Fisher Scientific, Waltham, MA, USA). Then, the entire genomic DNA was sequenced on Illumina Miseq Platform. The quality of raw data (300 bp each, paired-end reads) was assessed using FastQC 0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc ) and then were filtered for quality and trimmed using Skewer v0.2.2 (Jiang et al., 2013 (link)). Finally, 2 GB clean data was assembled using Geneious 11.1.5 (Kearse et al., 2012 ) based on obtained mt cox1 and rrnS gene sequences. We assembled all minichromosomes individually in full length as described previously (Fu et al., 2020a (link)). The gene borders were predicted with MITOS web serves (http://mitos.bioinf.uni-leipzig.de/index.py ) and manually curated. The location of protein-coding genes was further confirmed with BLAST searches of the NCBI database. tRNA genes were further confirmed using ARWEN (Laslett and Canbäck, 2008 ) and the program tRNAscan-SE (Lowe and Chan, 2016 (link)). The rRNA genes (rrnL and rrnS) were further confirmed by the boundary of the adjacent tRNA genes. The entire fragmented mt genome maps were produced using Microsoft Powerpoint.
7
Whole Genome Sequencing and Variant Calling
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Genomic DNA (gDNA) was extracted from strains of interest using Wizard genomic purification kit (Promega, Madison, WI). Genomic DNA was then quantified using the Qubit system (Thermo Fisher, Waltham, MA). Libraries were then produced using a Nextera XT Kit (Illumina, San Diego, CA). Quality of the libraries was controlled by Fragment Analyzer AATI (Agilent, Santa Clara, CA) and sequencing was performed using MiSeq Reagent Kits v2 on a MiSeq system (Illumina). Obtained reads were assembled with spades v. 3.11.1 (46 (link)) and mapped on reference genomes with bwa v. 0.7.17 (47 (link)). Variant calling was done with gatk 4.0.2.0 (48 (link)). Only variants supported by a minimum of 75% of the reads were considered, and the minimum sequencing depth to call a variant was set to 10. Identified SNPs were manually checked by visualizing the mapping with JBrowse (49 (link)).
8
Single-cell RNA-seq library preparation
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RNA-seq libraries were prepared using NEBNext® Single Cell/Low Input RNA Library Preparation Kit (New England Biolabs, Ipswich, MA, USA). Library preparation was initiated with 10 ng of total RNA. The RNA, along with all the other reagents, was thawed on ice. Further steps were conducted according to the manufacturer’s protocol until the final library amplification. Each final library was then purified using the MinElute PCR Purification Kit (Qiagen, Hilden, Germany) and then quantified on a Qubit system (Thermo Fisher Scientific). Multiplexed libraries were pooled in approximately equimolar ratios and were then run in a 2% agarose gel. The DNA between 300 and 700 base pairs was then cut and purified using the Qiagen Gel Extraction Kit (Qiagen). Library sequencing was performed at the RNomics platform of the Université de Sherbrooke (Sherbrooke, QC, Canada). Single-end sequencing was performed on all the samples with a 75 bp sequencing depth on a NextSeq machine. The raw data have been deposited at the Gene Expression Omnibus (GEO) under the subseries entry GSE218637.
9
Quantitative Proteomics of NCI-H-358 Cells
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NCI-H-358 cells were obtained from ATCC (Catalog #5807) and were reconstituted and passaged according to the included instructions using 6 well culture dishes. For bulk cell experiments, cells were aspirated, washed with ice-cold water which was rapidly aspirated prior to addition of S-Trap lysis buffer. All steps of the S-Trap mini protocol were performed according to manufacturer instructions (ProtiFi) with the exception that alkylation and reduction were not performed. Peptides for spectral library generation were labeled with the 128 °C reagent from the TMTPro reagents according to all manufacturer protocols. The peptides were fractionated by using high pH reversed-phase spin columns (Pierce) and eluted peptides were centrifuged to near dryness prior by SpeedVac. Peptides for use as carrier channels were labeled with the 134 N reagent from the TMTPro, lyophilized, and the concentration was determined using a Qubit system (Thermo Fisher) following manufacturer instructions.
10
RNA Quality Assessment for Sequencing
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Using 20 µL preparations, each, of DNA-free RNA from 37 patients, the RNA quality number (RQN) for each RNA preparation was determined using the Qubit system (Thermo Fisher) and by electrophoretic separation. While an RQN > 7 was considered suitable for sequencing, slightly lower RQNs (minimum: 6.2) were considered sufficient in the absence of degradation evidenced by chromatography. Subsequently, next-generation sequencing was performed.
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