Nextera xt

Viral RNA was prepared for next-generation sequencing using the Nextera XT library preparation kit (New England BioLabs, Ipswich, MA) following the manufacturer’s instructions. Briefly, viral RNA was quantified, with amounts expressed as genome equivalents (GE/ml), treated with RQ1 DNase (Promega) and used as templates in reverse transcription reactions performed using Superscript IV (ThermoFisher) and random 15-mer primers. Reaction products were treated with RNase H and amplification using the NEBNext® Ultra™ II Q5® Master Mix (New England Biolabs) and random 15-mer primers. Amplified products were fragmented and adapters and indexes added using Nextera XT (Ilumina) with in house Nextera XT dual indexes per manufacturer protocol. The index libraries were then amplified with a KAPA Real-time Library Amplification Kit (KAPA BioSystems, Indianapolis, IN) and purified using Ampure XP beads (Beckman). Final libraries were pooled by Qubit (Invitrogen) concentration and analyzed for size distribution using the Agilent High Sensitivity D1000 Screen Tape on an Agilent Tapestation 2200. Final quantification was performed using the NEBNext® Library Quant Kit for Illumina® (New England Biolabs), and 300 nt pair-end reads were generated using the Illumina MiSeq at the CSU Next-Generation Sequencing Facility.

Libraries for the fgf20a^−/− experiment were prepared with the Ovation RNA-Seq System V2 (7102, NuGEN) for cDNA amplification, followed by NexteraXT (Illumina) for library preparation. These libraries were sequenced on the HiSeq 2000 (Illumina), with paired-end 75 bp reads. Libraries for the constitutive active Fgfr1 experiment were prepared with the Clontech SMARTer kit (634926, TaKaRa) for cDNA amplification, followed by NexteraXT (Illumina) for library preparation. These libraries were sequenced on the HiSeq 4000 (Illumina), with single-ended 75 bp reads.

DNA concentrations in extracts were measured on the Qubit double-stranded DNA (dsDNA) HS assay kit (Invitrogen). Libraries for paired-end sequencing were constructed from DNA extracts ranging from < 50 ng/ml to 0.2 ng/µl, using the Illumina NexteraXT (Illumina, California, USA) Guide 150319425031942 and following protocol revision E. The Pooled NexteraXT libraries were loaded onto an Illumina MiSeq reagent cartridge using MiSeq reagent kit v3 and 500 cycles with a standard flow cell.

Genomic DNA was extracted from pure bacterial cultures using the Epicentre MasterPure Complete DNA and RNA purification kit (Epicentre, Madison, WI) per the manufacturer’s instructions. Genomic DNA was then quantified using a Qubit 2.0 fluorometer (Life Technologies, Inc., Carlsbad, CA) and diluted to 0.2 ng/µl for Illumina Nextera XT (FC-131-1096) (Illumina, Inc., San Diego, CA) DNA library preparation. Genomic tagmentation, PCR of tagged DNA, and PCR product cleanup were done according to the manufacturer’s instructions. The Qubit 2.0 fluorometer and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with the high-sensitivity DNA kit were both used for library dilution to 4 nM for loading into an Illumina MiSeq sequencer, which generated 250-bp reads. Demultiplexing was performed as previously described (13 (link)). Leech control isolate Hm21 was sequenced in a different study as previously described (10 (link)), and leech-derived isolate Hv13-B-10d was sequenced using PacBio RS II.

Control, non-injured Tnmd^−/−ScxGFP⁺ and WT ScxGFP⁺ Achilles tendons (n = 2) were explanted and GFP⁺ cells were isolated by collagenase digestion (8 h) and filtration according to [22 (link)]. Next, cells (n = 20/genotype) were resuspended in PBS, placed on Adcell diagnostic slides (Thermo Fisher, Waltham, Massachusetts, USA), picked up in 1 µl PBS each using a micromanipulator (Patchman NP2) with pump (CellTram, both Eppendorf, Hamburg, Germany) and subsequently stored in Smart-Seq2 lysis buffer at −80 °C. The whole transcriptome amplification (WTA) and Illumina Nextera XT library preparation (Illumina, San Diego, California, USA) were performed as described by Picelli et al. [32 (link)]. The libraries were quantified using the KAPA Library Quantification kit (Roche Diagnostics, Mannheim, Germany), pooled in equimolar amounts and sequenced paired-end with read length of 2 × 150 bp and yield of 30 million reads per library on an Illumina HiSeq. In total, six ScxGFP⁺ cells (n = 6/genotype) were subjected to scRNA-Seq. Bioinformatic analysis is described in Supplementary information.

VPRI specimen 18536 was used as a DNA representative from each of the 13 DNA extraction protocols, in a comparison study of two library preparation kits, Illumina Nextera XT^® (New England Biolabs) and NuGen Ovation^® Ultralow System V2 (NuGen).
Illumina Nextera XT^® double indexed and NuGen Ovation^® single indexed sequencing library preparations were completed for 13 VPRI 18536 DNA samples as per manufacturer’s instructions (S1 File). No DNA repair was performed on the fungarium DNA samples. The NuGen Ovation^® Ultralow System V2 libraries DNA samples were fragmented to 350 bp by sonication using Covaris S-Series Focused ultrasonicator. Fragmentation sonication settings are shown in S2 File. DNA library concentrations were quantified using Promega Quantus^™ fluorometer and Agilent 2200 TapeStation^®. The finalised Illumina Nextera XT^® and NuGen Ovation^® Ultralow System V2 libraries were paired-end sequenced on the Illumina^® HiSeq 3000 platform. Except for DneP+ Illumina Nextera XT^® and NuGen Ovation^® Ultralow System V2 libraries which were sequenced on Illumina^® MiSeq using the reagent V3 600 cycles kit due to a changeover in sequencing platforms in our facility and Illumina^® HiSeq 3000 is no longer available.