Agilent 2100 bioanalyzertm

All flash frozen leaf samples were ground using a mortar and pestle in liquid nitrogen. RNA was extracted from individual samples using the Qiagen® RNeasy® Plant Mini Kit (Qiagen®, Germantown, MD) with some modifications to the manufacturer’s protocol. Samples were incubated at 56 °C for two minutes and vortexed occasionally to disrupt tissue. Additionally, three rounds of RPE buffer washes were done instead of two. RNA samples were DNase treated using the Ambion® TURBO DNA-free kitTM (Ambion®, Austin, TX) to remove genomic DNA contamination. DNase-treated RNA samples were cleaned with Qiagen® RNeasy® MiniElute Cleanup Kit (Qiagen®, Germantown, MD). To ensure the integrity of RNA samples, all samples were checked using an Agilent® 2100 BioanalyzerTM (Agilent®, Santa Clara, CA) and samples with RNA Integrity Number (RIN) of ≥6.9 considered to be of good quality for RNA-seq. RNA from three leaflets (obtained from three different plants) was pooled in equal amounts for each RNA pool to be analyzed. A total of 48 RNA pools (3 replicates × 2 time points × 2 treatments × 4 soybean genotypes) were submitted to the DNA facility at Iowa State University for multiplex library preparation and single end sequencing using the Illumina HiSeq 2500 instrument. Each of the 48 cDNA libraries was sequenced at a read length of 100 base pairs.

The DNA from plasma and from histone-containing NPC was isolated using the “DNA Isolation Kit” (BioSilica Ltd., Novosibirsk, Russia) according to the manufacturer’s protocols and concentrated by precipitation in acetone as triethylammonium salts: glycogen (5 μL) (Fermentas) and 50 mM trimethylamine (10 μL) were added to the sample (100 μL) and precipitated with acetone (500 μL). After incubation for 15 h at −20 °C, the DNA was precipitated by centrifugation at 17,000× g for 20 min at 4 °C and the precipitate was dissolved in H₂O (12 μL). The concentration of isolated DNA was measured by quantitative polymerase chain reaction (Q-PCR) specific for long interspersed nuclear element 1 (LINE-1) repetitive elements [14 (link)]. The Q-PCR was performed with an ICycler iQ5 (Bio-Rad, Hercules, CA, USA) as described earlier [14 (link)]. Genomic DNA from human leukocytes served as a standard for obtaining the calibration curves. The DNA concentration was estimated according to the initial volume of each blood sample.
The size of NPC DNA was evaluated using a “High Sensitivity DNA Kit” and Agilent 2100 Bioanalyzer^TM (Agilent Technologies, Waldbronn, Germany) in SB RAS Genomics Core Facility (ICBFM SB RAS, Novosibirsk, Russia).

Flash frozen tissue was ground in liquid nitrogen with a mortar and pestle. RNA was extracted using a Qiagen® RNeasy® Plant Mini Kit (Qiagen®, Germantown, MD). The manufacturer’s protocol was followed using ~300 mg of ground tissue which was lysed using the RLT buffer and tubes were incubated at 56°C for two minutes with 800 rpm shaking to aid in tissue disruption. RNA was treated with an Ambion® TURBO DNA-free™ kit (Ambion®, Austin, TX) to remove all contaminating DNA. RNA quality was analyzed using an Agilent® 2100 Bioanalyzer TM (Agilent®, Santa Clara, CA). RNA was considered to be of good quality if the RNA was not degraded or was only marginally degraded. Equal amounts of RNA from three plants were pooled for each biological replicate prior to sequencing. In addition, the same RNA samples were used by Atwood et al. [11 (link)] to measure differential gene expression of Replication Protein A subunits by quantitative reverse transcription polymerase chain reaction.

Flash frozen tissue was ground in liquid nitrogen with a mortar and pestle. RNA was extracted using a Qiagen^® RNeasy^® Plant Mini Kit (Qiagen^®, Germantown, MD, USA). The manufacturer’s protocol was followed using ~300 mg of ground tissue which was lysed using the RLT buffer and tubes were incubated at 56 °C for two min with 800 rpm shaking to aid in tissue disruption. RNA was treated with an Ambion^® TURBO DNA-free^TM kit (Ambion^®, Austin, TX, USA) to remove all contaminating DNA. RNA quality was analyzed using an Agilent^® 2100 Bioanalyzer TM (Agilent^®, Santa Clara, CA, USA). RNA was considered to be of good quality if the RNA integrity number (RIN) was greater than 7. Of the four biological replicates for each sample, the three replicates with the highest RIN values were selected for RNA-seq analyses.