Agilent rna 6000 pico kit

Total RNAs were isolated using the Isogen II (NipponGene). The sizes of cellular and exosomal RNAs collected from the PK-45H cells were examined using the Agilent 2100 Bioanalyzer and Agilent RNA 6000 Pico Kits (Agilent Technologies)^{36 (link)}. Total RNAs from HUVECs treated with exosomes were subjected to microarray analysis, as described below.

RNA sequencing was conducted on three C. albicans isolates (CA1, CA2, CA3) at three time points during biofilm formation (24 h, 48 h, 72 h). The removal of ribosomal RNA and the isolation of poly(A) RNA from the total RNA samples were performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s protocol. Libraries for sequencing were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s manual. The quality control of the isolated RNA and prepared libraries was performed using an Agilent 2100 Bioanalyzer with an Agilent RNA 6000 Pico Kit and Agilent High Sensitivity DNA Kit (all Agilent Technologies, Santa Clara, CA, USA), respectively. Sequencing was carried out using the HiSeq4000 platform (300 cycles mode) and the reagents included in the HiSeq 3000/4000 SBS Kit (both Illumina, San Diego, CA, USA). The experiments were performed in duplicate, giving a total of 18 sequenced samples (three isolates at three time points in duplicates). The average number of raw reads obtained in the studied samples was 21,569,234 (Figure S3).

The messenger RNA from chick skeletal muscle cells was extracted and isolated by Dynabeads mRNA DIRECT Micro Kit (Invitrogen). Samples were pooled from three biological replicates each. The library preparation was carried out using the Ion Total RNA-Seq Kit v2 (Life Technologies) with 500 ng of PolyA (RNA) according to manufacturer’s instructions. To assess the yield and size distribution of the fragmented RNA we used Qubit RNA Assay Kit (Invitrogen) and Agilent RNA 6000 Pico Kit (Agilent, GE). The complementary DNA (cDNA) was amplified without barcoding. The dsDNA HS Assay Kit (Invitrogen) and Agilent High Sensitivity DNA Kit (Agilent, GE) were used to assess the yield and size distribution of the amplified DNA. The template was made in One Touch System by Ion One Touch 200 template kit V2 (Life technologies) according to manufacturer’s instructions. The transcriptome sequencing was performed using Ion PGM 200 Sequencing Kit (Life Technologies) with 318 chips in Ion Torrent PGM machine.

cDNA libraries were generated using the SMARTer stranded total RNA-Seq kit (pico input mammalian) (Cat No. 635007) according to the manufacturer's instructions with the exception of replacing the reverse primer with an N6-oligo primer lacking the oligo-dT primer. RNA denaturation time for plasma samples was 4 min for all samples. Final PCR amplification was done for 16 cycles. Ribosomal cDNA was removed using ZapR treatment with R-probes. Purification was done with Agencourt AMPure XP beads (A63881, 60 ml) (Beckman Coulter). Following Agilent Bioanalyzer profiling (Agilent RNA 6000 Pico kit) (Cat. No 5067-1513), cDNA libraries were pooled in equimolar amounts.

RNA extraction was done with a RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored at −80 °C until use. DNA residues were eliminated by DNase I treatment using a DNA-free kit (Ambion, Austin, TX, USA). Enrichment of mRNA was performed using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Further, cDNA libraries were prepared with a NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). Qualitative and quantitative evaluation of the RNA and cDNA libraries was performed with a Qubit fluorometer (Invitrogen, USA) and 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) using the Agilent RNA 6000 Pico Kit (Agilent Technologies). Sequencing was performed on the HiSeq2500 platform (Illumina, San Diego, CA, USA) using single read sequencing at the Joint KFU–Riken Laboratory, Kazan Federal University (Kazan, Russia).

HSPCs in the BM, MSCs, and OLCs in the BF were sorted directly into 800 µl TRIzol Reagent (15596018, Thermo Fisher Scientific) and stored at −80 °C until thawed for RNA extraction. Total RNA was isolated following standard protocol. GenElute LPA (56575, Sigma Aldrich) was used as a carrier for ethanol precipitation. After extraction, the RNA pellet was resuspended in 7.5 µl of DEPC-treated water (AM9916, Thermo Fisher Scientific). The quality and quantity of total RNA were examined on a 2100 Bioanalyzer (Agilent) using Agilent RNA 6000 Pico Kit (5067-1513, Agilent).