Agentcourt ampure xp beads

The genomic DNA was extracted from all the rumen samples employing PSP Spin Stool DNA Plus Kit (Invitek, Berlin, Germany) using the protocol of Dollive et al. (2012) (link). The genomic DNA was amplified using the specific primers (27F) and BSR357, targeting the V1–V2 region of the 16S rRNA bacterial gene. The primer sequences and PCR conditions for Roche 454 are described in Pitta et al. (2014) (link). Though the primer sequences for Ion Torrent were similar to Roche 454, the forward primer carried the Ion Torrent trP1 (5′-CCTCTCTATGGGCAGTCGGTGAT-3′) and the reverse primer carried the A adapter (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′), followed by a 10–12 nucleotide sample-specific barcode sequence and a GAT barcode adapter. The PCR mix was prepared using the Platinum PCR SuperMix High Fidelity kit (Invitrogen, Carlsbad, CA, USA). PCR conditions were the same for Roche 454 and Ion Torrent, as given by Pitta et al. (2014) (link). Amplicons of 16S rDNA were purified using 1:1 volume of Agentcourt AmPure XP beads (Beckman-Coulter, Brea, CA, USA). The purified PCR products from the rumen samples were pooled in equal concentration prior to sequencing in Roche 454 (Roche 454 Life Sciences, Branford, CT, USA) and Ion Torrent platforms.

A custom, single pool, multiplexed, PCR-based, NGS library panel was designed using Ion AmpliSeq designer software (Thermo Fisher Scientific, Waltham, MA, USA) to target 97 specific regions from 94 genes for a total panel size of 15.58 Kb per library^{34 (link)}. The information on the genomic coordinates of the amplicons included in the panel can be found in Supplementary Table 1. The Libraries were constructed using Ion AmpliSeq^TM Library Kit v2.0 (Life Technologies), according to the manufacturer’s instructions^{34 (link)}. To simply explain, 10 ng of DNA from either frozen, non-WGA FFPE or FFPE WGA products were amplified for 21 cycles, followed by FUPA treatment and ligation of Ion Xpress plus Universal adaptors. The libraries were purified with Agentcourt Ampure XP beads (Beckman Coulter, Fullerton, CA) and re-suspended in 1× low TE buffer. The libraries were quantified by using the Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific; Cat. No. 4468802).

The JAK2 p.V617F mutation was excluded [18 (link)]. JAK2 exon 12 was PCR-amplified with flanking primers. Two distinct gel bands on a 2 % agarose gel were excised, purified by MinElute Gel extraction kit (QIAGEN, Hilden, Germany) and sequenced. Amplicons of MEFV exons 1–10 (LRG_190t1), NLRP3 exon 3 (LRG_197t1), TNFRSF1A exons 2–4 (LRG_193t1), and MVK exons 2–11 (LRG_156t1) were sequenced after Exo-Sap treatment. Sequencing reactions were performed using the Big Dye Terminator kit (Applied Biosystems, California, USA) on an ABI 3130XL automated sequencer, and the sequences were analyzed with BioEdit or blast software.
For targeted deep sequencing, a first PCR reaction surrounding the c.1955 nucleotide change of MEFV was performed in triplicate. Barcodes were incorporated to amplicons during a second, nested round of amplification. Amplicons were then purified using Agentcourt AMPureXP beads (Beckman coulter, Nyon, Switzerland) and the DNA quantified using High Sensitivity DNA kits for Bioanalyzer (Agilent technologies, Ca, USA). Sequencing and analyses were performed using the 400 bp kit on a PGM system, Ion Torrent server and Ion Reporter software, according to manufacturer’s instructions (Ion Torrent, Life Technologies, Guilford, Connecticut, USA). Coverage at the c.1955 position was at least ×3000.

KPC cells expressing doxycycline-inducible shRNA targeting CTR9, CDC73, MYC or luciferase as a control were incubated with doxycycline (1 mg/ml) for 48 h. RNA was isolated using peqGOLD TriFast (PeqLab). Total RNA was extracted using the miRNeasy kit (QIAGEN) including on-column DNase I digestion. mRNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and library preparation was performed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina according to the instructions. Libraries were size selected using Agentcourt AMPure XP Beads (Beckman Coulter), followed by amplification with 11 cycles of PCR.