All enrolled patients underwent targeted sequencing to evaluate the presence of CH mutations. DNA was extracted from whole blood using the QIAGEN Mini Kits (catalog no. 27104) according to the manufacturer’s recommendations. We batch-sequenced samples using a custom-made capture panel designed to tile known CH genes on an Illumina NovaSeq 6000. Somatic mutations were called using publicly available methods in workflow description language in Mutect2 on the Broad Institute’s Terra Platform (https://terra.bio/ ). A putative variant list was formulated and then cross-referenced with a list of known CH driver mutations. Variants were then filtered for read quality, including sequencing depth and minimum alternate allele read depth. Sex was determined based on self-report and findings are applicable to both sexes. There were 8 women and 15 men in this study. Population-level studies of CH do not indicate a sex-specific effect; thus, the study design was not powered to pursue these analyses. Patient mean age was 72 at the time of sampling. CHIP mutations were determined via targeted capture panel, resulting in eight TET2 and six control patients. There were no treatments as a part of this study.
Mini kits
Manufactured by Qiagen
Sourced in Germany, United States
Mini Kits are compact, easy-to-use lab equipment designed for efficient nucleic acid extraction and purification. They provide a streamlined workflow for small-scale sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy micro, by Qiagen (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nebnext 2x master mix, by New England Biolabs (2 mentions)
Spri bead, by Beckman Coulter (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (1 mentions)
Fugene, by Promega (1 mentions)
Mint universal cdna synthesis kit, by Evrogen (1 mentions)
Trimmer cdna normalization kit, by Evrogen (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Qiagen (1 mentions)
Rnaeasy micro, by Qiagen (1 mentions)
Superscript first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
17 protocols using mini kits
1
Targeted Sequencing for Clonal Hematopoiesis
2
Plasmid Transformation and Purification
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Plasmids were heat shock transformed into DH5α competent E. coli and selected for using antibiotics (puromycin to select for pBABEpuro ErbB2; zeocin to select for p85-GFP, kanamycin to select for Gab1-wt; and ampicillin to select for Gab1delta). Plasmids were prepared using Sigma Aldrich midi kits (Sigma Aldrich, St Louis, MO) or Qiagen mini kits (Qiagen, Valencia, CA) and transfected via Fugene (Promega, Madison, WI). Plasmids were sent out for sequencing confirmation (TCAG, Sick Kids, Toronto, On)
3
RNA Extraction and Transcriptome Sequencing
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Total RNA was extracted from 30 mg of liver or white skeletal muscle using the RNeasy tissue and RNeasy fibrous tissue, respectively, mini kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration and purity was determined spectrophotometrically at 260/280 nm using Nanodrop ND-1000 (Thermo Fischer Scientific, Waltham, MA, USA). RNA integrity was determined with an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only samples with RNA Integrity Number (RIN) > 9.2 were used for subsequent studies. Pools consisting of 1 μg of total RNA isolated from 6 individuals per condition (fasting and feeding with diets HLL, MHL, MLH, LHH and LLH; 36 fish and RNA samples in total) were used to construct liver and white skeletal muscle cDNA libraries. The dsDNA synthesis was performed using a MINT-Universal cDNA synthesis kit (Evrogen, Moscow, Russia). To increase the presence of rare transcripts, the cDNAs libraries were normalised using TRIMMER cDNA normalization kit (Evrogen, Moscow, Russia) following manufacturer’s instructions. Sequencing of cDNAs libraries was performed using a GS FLX 454 platform (Roche, Basel, Switzerland) at the CCiTUB of the Universitat de Barcelona (Barcelona, Spain).
4
CRISPR-based Modification of DBT1 Loci
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Genomic DNA was extracted from cell pellets using the QIAGEN (QIAGEN, Hilden, Germany) Midi or Mini Kits based on the size of the cell pellet (51183, 51104) according to the manufacturer’s recommendations. DBT1 loci were first amplified with 17 cycles of PCR using a touchdown protocol and the NEBnext 2x master mix (New England Biolabs M0541). The resulting product served as input to a second PCR, using primers that appended a sample-specific barcode and the necessary adaptors for Illumina sequencing. The resulting DNA was pooled, purified with SPRI beads (A63880, Beckman Coulter, Brea, CA), and sequenced on an Illumina MiSeq with a 300-nucleotide single-end read with an eight nucleotide index read. For each sample, the number of reads exactly matching the wild-type and edited DBT1 sequence were determined (Figure 5—source data 1 ).
5
Quantitative Gene Expression Analysis
6
Quantitative PCR Analysis of Gene Expression
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qPCR analysis was performed with the QuantStudio 6 Flex Real‐Time PCR System (Applied Biosystems, CA). Total RNA was extracted with RNAeasy Micro (cells), Trizol, and/or Mini Kits (tissues) (Qiagen, CA). One thousand nanogram total RNA from each of the pooled PVC cells/tissues or atrial tissue samples was reverse transcribed with SuperScriptTM First Strand Synthesis System for RT‐PCR (Invitrogen, CA). Real‐time PCR was performed in triplicates for every sample using primers listed in Table 1 . Using SYBR Green/ROX probe (Thermo Fisher, MA), averaged Ct values of each qPCR reaction from the target gene were normalized with the average Ct values of the housekeeping gene 18S, which ran in the same reaction plate to obtain the ∆Ct value (Barajas‐Martinez et al. 2017 ; Goodrow et al. 2017 ). Expression was normalized from ∆Ct values for each gene against reference housekeeping gene 18S, using the formula 2−∆∆Ct (1 × 106) (Livak and Schmittgen 2001 ):
7
RNA Isolation and RNA-seq Analysis
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RNA was isolated from cells with Qiagen RNeasy Micro or Mini Kits (on the basis of cell number), according to the manufacturer’s instructions. RNA-seq was done with an Illumina HiSeq2500 instrument at Genewiz corp., Suzhou, China.
8
Total RNA Extraction and qPCR Analysis
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Total RNA was extracted using RNeasy Lipid or Mini Kits (Qiagen Inc., Valencia, CA, USA) for tissue and cell culture samples, respectively, following the supplier’s instructions. RNA content was quantified using NanoDrop technology (Fisher Scientific SAS, Illkirch Cedex, France) and quality checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA samples were treated with DNase Treatment and Removal Reagents (Thermo Fisher, Waltham, MA, USA) and reverse-transcribed for 1 h at 37 °C with 150-ng random primers, 0.5-mM dNTPs, 20 units of RNAsin Ribonuclease Inhibitor and 200 units of M-MLV RT (Promega, Madison, WI, USA). Primer sequences and amplification conditions are reported in Table 2 . Real-time PCR was carried out with Platinum® SYBR® Green qPCR SuperMix-UDG (Thermo Fisher) on a DNA Engine OpticonTM 2 Continuous Fluorescence Detection System (MJ Research, Waltham, MA, USA). All experiments were performed in duplicate. Samples (5 ng of cDNA) were normalized by 18S rRNA content and reported as an arbitrary unit (a.u.) ratio or as a fold increase/decrease with respect to the control.
9
Targeted DNA Sequencing of TOP2A/B
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Genomic DNA was extracted from cell pellets using the QIAGEN (QIAGEN, Hilden, Germany) Midi or Mini Kits based on the size of the cell pellet (cat # 51183, 51104) according to the manufacturer’s recommendations. TOP2A and B loci were first amplified with 17 cycles of PCR using a touchdown protocol and the NEBnext 2x master mix (New England Biolabs M0541). The resulting product served as input to a second PCR, using primers that appended a sample-specific barcode and the necessary adaptors for Illumina sequencing. The resulting DNA was pooled, purified with SPRI beads (A63880, Beckman Coulter, Brea, CA), and sequenced on an Illumina MiSeq with a 300 nucleotide single-end read with an eight nucleotide index read. For each sample, the number of reads exactly matching the wild-type and edited TOP2A/B sequence were determined (S13 Table ). The numbers of reads were compared using a Fisher’s exact test.
10
Quantitative Gene Expression Analysis
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Adipose tissue, brain, intestinal mucosal scrapings and spleen RNA was extracted using Qiagen Lipid Mini kits and liver RNA was isolated using Qiagen Mini kits according to manufacturer's instructions. RNA was quantified and checked for purity using the Nanodrop spectrophotometer (Nanodrop 1000, Wilmington, DE). cDNA was generated from 1 μg of RNA, and real-time quantitative PCR was performed using SYBR Green (Applied Biosystems 7300, Carlsbad, CA). Fold-changes were calculated as 2–ΔΔCT, with cyclophilin B used as the endogenous control. Primer sequences are listed in Table S1 .
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