Minelute spin column

RNA extraction was carried out using the RNeasy Plus Micro kit, together with gDNA eliminator and MinElute spin columns (Qiagen, United Kingdom). cDNA synthesis (mRNA) was performed using the RT2 First Strand kit (Qiagen, Netherlands) according to the manufacturer’s protocol. qPCR analysis was performed using the Stratagene MX3000P RT-PCR System (Stratagene, La Jolla, CA, United States) in a 25−μL reaction mixture. Expression relative to housekeeper Rplp1 was calculated as ΔCt.

The genome for this strain was obtained at the ultrasequencing core facility of the CRG, using Illumina GAIIx sequencing machine. DNA was fragmented by nebulization or in Covaris to a size approximately 300 bp. After shearing, the ends of DNA fragments were blunted with T4 DNA polymerase and Klenow fragment (New England Biolabs). Then, DNA was purified with a QIAquick polymerase chain reaction (PCR) purification kit (Qiagen). Thereafter, 3′-adenylation was performed by incubation with dATP and 3′–5′-exo-Klenow fragment (New England Biolabs). DNA was purified using MinElute spin columns (Qiagen), and double-stranded Illumina paired-end adapters were ligated to the DNA using rapid T4 DNA ligase (New England Biolabs). After another purification step, adapter-ligated fragments were enriched, and adapters were extended by selective amplification in an 18-cycle PCR reaction using Phusion DNA polymerase (Finnzymes). Libraries were quantified and loaded into Illumina flow cells at concentrations of 7–20 pM. Cluster generation was performed in an Illumina cluster station. Sequence runs of 2 × 76 cycles were performed on the sequencing instrument. Base calling was performed using Illumina pipeline software. In multiplexed libraries, we used 4-bp internal indices (5′-indexed sequences). Deconvolution was performed using the CASAVA software (Illumina).

Fresh unused cartomizers were dissected to separate the fibers from the atomizing unit, as described previously [13 (link)]. The white plug in the end of the mouthpiece was removed to reveal the fibers surrounding the atomizing unit. The inner and outer fibers were centrifuged in MinElute Spin columns (Qiagen, Valencia, CA) at 14,000 revolutions/minute for 4–6 minutes to separate the fluid from the fibers [13 (link)].

Cultured cells were crosslinked using 1% formaldehyde. Crosslinking was terminated by adding glycine to a final concentration of 0.125 m. Cells were scraped off the dish, collected into a fresh 1.5 mL tube, and resuspended in ChIP lysis buffer supplemented with proteinase inhibitor (Bimake, Shanghai, China). Chromatin was sheared into 200–1000 bp fragments by sonication under the proper conditions. IgG or ChIP degree antibodies were added to Protein A/G magnetic beads (Bimake, Shanghai, China) and rotated at room temperature. After 30 min, the chromatin mixture was added to the beads, and the sample was rotated at 4 °C overnight. Then, the tube was subjected to a magnetic field to remove the supernatant, which contained nonspecific fragments. The beads were washed 4 times and then eluted using MinElute Spin Columns (Qiagen, Hilden, Germany). The primers used in the ChIP assay are listed in Table S2 (Supporting Information).

For 7 bone and tusk powder samples, the standard extraction protocol was performed twice: once with QIAquick spin columns (as in the original protocol) and once with MinElute spin columns (both from Qiagen).

Genome-wide enrichment of double stranded libraries was performed through custom target enrichment kits (Arbor Bioscience). RNA baits with a length of 60 nucleotides and a 4 bp tiling density were designed based on three reference genomes: Nichols (CP004010.2), SS14 (CP000805.1), Fribourg-Blanc (CP003902). 500 ng library pools were enriched according to the manufacturer’s instructions. Captured libraries were amplified in 100 µl reactions containing 1 unit Herculase II Fusion DNA polymerase (Agilent), 1× Herculase II reaction buffer, 0.25 mM dNTPs, 0.4 mM primers IS5 and IS6^{81 (link)} and 15 µl library template, with the following thermal profile: initial denaturation at 95 °C for 2 min, 14 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 30 s, followed by a final elongation at 72 °C for 5 min. Captured libraries were purified with MinElute spin columns (QIAGEN) and quantified with a D1000 High Sensitivity ScreenTape on an Agilent 2200 TapeStation.