2.1 Primers and Oligonucleotides
Primes and oligonucleotides were ordered at IDT (Integrated DNA Technologies, BVBA, Leuven, Belgium) at 100 µM in IDTE Buffer pH 8.0 and with standard desalting purification. All primers [78]
and oligonucleotides are listed in the appendix.
2.2 Chemicals
The chemicals that were used in this work are listed in Table 1.
Table 1: Chemicals
Chemical (product name) Source
Agencourt AMPure XP Beads Beckman Coulter, Brea, CA, USA
Axiom water Affimetrix/Thermo Fisher Scientific, Santa Clara,
CA, USA
Buffer EB Qiagen GmbH, Hilden, Germany
dNTP Set 100 mM Solutions (dATP, dGTP, dCTP, dTTP)
ThermoFisher Scientific, Waltham, MA, USA EDTA disodium salt 2-hydrate AppliChem GmbH, Darmstadt, Germany ethanol absolute, p. A. A1613 AppliChem GmbH, Darmstadt, Germany Hydrochloric Acid 0.5 M/37% p.A. Carl Roth GmbH & Co. KG, Karlsruhe, Germany LiChrosolv Water For Chromatography, LC-MS
Grade
Merck Chemicals GmbH, Darmstadt, Germany Sodium hydroxide p. A. (pellets) Carl Roth GmbH & Co. KG, Karlsruhe, Germany SYBR Green I (10,000X, in DMSO) ThermoFisher Scientific, Waltham, MA, USA Tris(hydroxymethyl)aminomethane p.A. Merck Chemicals GmbH, Darmstadt, Germany
2.3 In-House Buffers and Solutions
Custom buffers and their components are listed in Table 2.
Table 2: Components of custom buffers and solutions
Buffer/solution Components/recipe Amount
0.5 M Na2EDTA EDTA disodium salt 2-hydrate aqua dest
2 Materials
2.4 Enzymes and Enzyme Reaction Buffers
Commercial enzymes and components for enzymatic reactions are listed in Table 3.
Table 3: Commercial enzymes and enzyme reaction buffers
Enzyme/reaction buffer (product name) Source
10X Ampligase Reaction Buffer Epicentre, Madison, WI, USA Ampligase DNA Ligase (100 U/µL) Epicentre, Madison, WI, USA
Exonuclease I (E. coli) 20,000 U/mL New England Biolabs Inc., Ipswich, MA, USA Exonuclease III (E. coli) 100,000 U/mL New England Biolabs Inc., Ipswich, MA, USA
Hemo KlenTaq New England Biolabs Inc., Ipswich, MA, USA
iProof HF Master Mix Bio-Rad Laboratories, Hercules, CA, USA T4 DNA Ligase Reaction Buffer New England Biolabs Inc., Ipswich, MA, USA T4 Polynucleotide Kinase (10,000 Units/mL) New England Biolabs Inc., Ipswich, MA, USA
2.5 Consumables
The consumables that were used in this work are listed in Table 4.
Table 4: Consumables
Consumables (product name) Source
12.5 µL Matrix Tips Matrix/ThermoFisher Scientific, Hudson, NH,
USA
25 mL Disposable Multichannel Pipette Reservoirs Integra Biosciences AG, Zizers, Switzerland 3M™ Empore™ Sealing Tape Pad 3M, St. Paul, MN, USA
ABgene Storage Plate, 96-well (AB-0932) ABgene/ThermoFisher Scientific, Waltham, MA, USA
Adhesive PCR Plate Seals (AB-0558) ThermoFisher Scientific, Waltham, MA, USA CELLSTAR Serological Pipettes 2 mL, 5 mL, 10 mL,
25 mL
Greiner Bio-One GmbH, Frickenhausen, Germany
DNA LoBind Tubes, 1.5 mL/2.0 mL/5.0 mL, PCR clean
Eppendorf AG, Hamburg, Germany Falcon 15 mL/50 mL High Clarity PP Centrifuge
Tube, Conical Tube
Corning Incorporated (USA), Corning, NY, USA KIMTECH SCIENCE Purple Nitrile (gloves) Kimberly-Clark Worldwide, Inc., Dallas, TX, USA
Kimwipes Lite Kimberly-Clark Worldwide, Inc., Dallas, TX, USA
Millipak-20 Filter Unit 0.22 µm Merck Chemicals GmbH, Darmstadt, Germany SR-L10F, SR-L200F, SR-L1000F, SS-L10, SS-L250,
SS-L1000 pipette tips
Rainin Instruments, LLC/Mettler Toledo, Oakland, CA, USA
Stripe Tubes and Caps, 0.1 mL (qPCR) Qiagen GmbH, Hilden, Germany
Thermo-Fast 96 PCR Plate Non-Skirted (AB-0600) ABgene/ThermoFisher Scientific, Waltham, MA, USA
2.6 DNA Quantification and Analysis Kits
2.6 DNA Quantification and Analysis Kits
Commercial DNA quantification and analysis kits are listed in Table 5.
Table 5: Commercial DNA quantification and analysis kits
Kit (product name) Source
Agilent DNA 1000 Kit Agilent Technologies, Inc., Santa Clara, CA, USA Qubit dsDNA HS Assay Kit ThermoFisher Scientific, Waltham, MA, USA
2.7 Equipment and Machines
The equipment and machines that were used in this work are listed in Table 6.
Table 6: Equipment and machines
Item (product name) Source
6 tube magnetic stand AB Sciex LLC, Foster City, CA, USA
accu-jet pro pipette controller Brand GmbH & Co. KG, Wertheim Main, Germany Agilent 2100 Bioanalyzer Agilent Technologies, Inc., Santa Clara, CA, USA
Centrifuge 5424 Eppendorf AG, Hamburg, Germany
DURAN laboratory flask 1 L, 5 L DURAN Group GmbH, Wertheim Main, Germany
EF 103 easy-fit Scotsman Ice Systems, Vernon Hills, IL, USA
Freezer (-20°C) Liebherr-International Deutschland GmbH, Biberach
an der Riß, Germany
Freezer (-20°C) Privileg/OTTO GmbH & Co. KG, Hamburg, Germany Linux Cluster (4 Dell PowerEdge R815 à 48
Cores, 256 GB RAM, Sun Grid Engine)
Max-Plank-Institut für Psychiatrie – Deutsche Forschungsanstalt für Psychiatrie, München, Germany
LKexv 2600 MediLine & FKS 2600 (fridge) Liebherr-International Deutschland GmbH, Biberach an der Riß, Germany
macro pipette controller Brand GmbH & Co. KG, Wertheim, Germany Matrix Electronic Multichannel Pipette Impact
2
Matrix/ThermoFisher Scientific, Hudson, NH, USA Mikro 20 (centrifuge) Andreas Hettich GmbH & Co. KG, Tuttlingen,
Germany
Milli-Q Synthesis (water purification system) Merck Chemicals GmbH, Darmstadt, Germany MS1, MS2, MS3, VF2 (vortexer) IKA-Werke GmbH & Co. KG, Staufen im Breisgau,
Germany peqSTAR 96 Universal Gradient, 96 HPL & 96X
(thermocycler)
VWR International, LLC, Radnor, PA, USA PerfectSpin P (plate centrifuge) VWR International, LLC, Radnor, PA, USA Qubit 2.0 Fluorometer Invitrogen, Carlsbad, CA, USA
Rainin Classic Pipette PR-2, PR-10, PR-20, PR-100, PR-200, PR-1000
Mettler-Toledo Rainin, LLC, Oakland, CA, USA Rainin Pipet-Lite XLS+ LTS L-2XLS, L-10XLS,
L-20XLS, L-200XLS, L-1000XLS, L12-10XLS, L12-20XLS & L12-200XLS
Mettler-Toledo Rainin, LLC, Oakland, CA, USA
Reax top & Reax 2 (vortexer) Heidolph Instruments GmbH & Co. KG, Schwabach, Germany
Refrigerated Centrifuge 4K15 Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany
2 Materials
continued table…
Item (product name) Source
Rotana 46 RS (centrifuge) Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany
Rotator 2-1175 neoLab Migge GmbH, Heidelberg, Germany
Rotofix 32 A (centrifuge) Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany
Rotor-Gene Q (qPCR thermocycler) QIAGEN GmbH, Hilden, Germany Scientific Computing Matrix (HPC-HA-Cluster,
Linux)
Helmholtz Zentrum München – Deutsches Forschungszentrum für Gesundheit und Umwelt, Neuherberg, Germany
Sigma 2-16 (centrifuge) Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany
Spectrafuge™ Mini Centrifuge Labnet International, Inc., Woodbridge, NJ, USA Sprout Mini Centrifuge 12V Heathrow Scientific LLC, Vernon Hills, IL, USA Titramax 100 (orbital shaker) Heidolph Instruments GmbH & Co. KG, Schwabach,
Germany