Qubit rna br

DNA, RNA and proteins were extracted from most of the experiments as shown in the experimental setup (Fig. 1). Only DNA was extracted from the monomer mineralization tests. Total DNA, RNA and proteins were extracted from all tests simultaneously by using the Quick-DNA/RNA Miniprep Plus Kit (Zymo Research, California, USA) according to the manufacturer’s instructions. To asses the integrity of isolated DNA and RNA, 5 μL of each sample was loaded in an agarose gel and visualized by using Intas Gel v. 0.2.14 software. To remove possible RNA contamination, the DNA samples were additionally treated with RNase A (AppliChem, Darmstadt, Germany) and purified by using DNA Clean and Concentrator kit (Zymo Research, California, USA) following manufacturer’s instructions. Quality of DNA and RNA was additionally analyzed via Nanodrop by using the Nanodrop 2000 v. 1.5 software (Nanodrop 2000, Thermo Fischer Scientific, Waltham, USA). The fractions of proteins isolated with Quick-DNA/RNA Miniprep Plus Kit (Zymo Research, California, USA) were further precipitated with acetone according to the manufacturer’s instructions.
Purified total DNA, RNA, and proteins were quantified to determine concentration by using Qubit RNA BR, dsDNA BR, and Protein Assay kits and measured on a Qubit 3.0 Fluorometer (Invitrogen, California, USA).

Frozen PBMCs were defrosted into RPMI media supplemented with 10% foetal calf serum. Total RNA was extracted from one million cells using a MagMAX mir Vana Total RNA extraction kit on a MagMAX™ Express-96 Deep Well Magnetic Particle Processor. Prior to sequencing library preparation, quality control was performed by RNA size analysis, on a 4200 Tapestation using RNA ScreenTape (Agilent Technologies, Santa Clara, USA). All samples exhibited RIN values ranging between 7.4 and 9.1 and were quantified using a Qubit RNA BR (ThermoFisher Scientific, Waltham, MA). Sequencing library preparation was performed using the Illumina Stranded mRNA Prep kit (Illumina, San Diego, USA) according to the manufacturer's instructions and automated using a Hamilton NGStar (Hamilton, Bonaduz, SW). Sequencing library quality was assessed using Tapestation 4200 (Agilent Technologies, Santa Clara, USA) and Qubit (ThermoFisher Scientific, Waltham, MA) before being diluted to 5 µM. Libraries were randomly split into nine pools of 8–10 samples. Pooled libraries were quantified using the NEBNext Illumina library quantitation kit (NEB, Ipswich, USA) and Qubit (ThermoFisher Scientific, Waltham, MA) and adjusted to 5 µM prior to spiking with 1% PhiX (Illumina, San Diego, USA). Pooled libraries were sequenced on two 2 × 75 cycle paired-end sequencing runs on a NextSeq 550 System (Illumina, San Diego, USA).

Total RNA was extracted from wildtype and H3.3 KO Tetrahymena during vegetative growth using the RNeasy extraction kit (Qiagen) following the manufacturer’s instructions. Two independent biological samples for each condition were generated. RNA was treated with DNase, and total RNA was quantified using Qubit RNA BR (cat # Q10211, Thermo Fisher Scientific Inc., Waltham, USA) fluorescent chemistry. 1000 ng per sample was processed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat # E7760L; New England Biolabs, Ipswich, USA; protocol v. v3.1_5/20), including PolyA selection. 1 uL top stock of each purified final library was analyzed on an Agilent Bioanalyzer dsDNA High Sensitivity chip (cat # 5067-4626, Agilent Technologies Inc., Santa Clara, USA). The libraries were quantified using the Quant-iT dsDNA high sensitivity (cat # Q33120, Thermo Fisher Scientific Inc., Waltham, USA) and were pooled at equimolar ratios after size adjustment. The quantified pool was hybridized at a final concentration of 2.215 pM, and single-end reads were obtained on an Illumina NextSeq 500 platform using a full High-Output v2.5 flowcell at 75 bp read lengths.