Agilent rna 6000 nano

DNA and RNA were extracted from MACS-sorted blasts using the Allprep mini kit and the robotic workstation QIAcube (Qiagen, Hilden, Germany). RNA quality was evaluated using the Agilent RNA 6000 Nano and Pico kits in the Agilent 2100 Bioanalyzer. RNA concentration was calculated using the Qubit™ RNA High Sensitivity and Broad Range kits, while DNA concentration was calculated using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).

Total RNA was quantified using NanoDrop ND-1000 spectrophotometer (Isogen Life Science, Temse, Belgium). RNA purity was assessed by checking the absorbance at 260 nm, 280 nm and 230 nm. An A260/A280 ratio <1.8 denotes protein contamination and an A260/A230 <2 indicates presence of phenol, chaotropic salts (guanidinium thiocyanate) or proteins. RNA integrity was analyzed with the Agilent RNA 6000 Nano or Pico kits (using 1 μL of RNA from the stock solution or from equally diluted samples) on the Agilent Bioanalyzer 2100 (Agilent technologies, Diegem, Belgium) and evaluated based on the RNA Integrity Number (RIN) that classifies the RNA as very degraded (RIN = 1) to intact (RIN = 10). The quantity and the profile of small RNAs were assessed by capillary electrophoresis and fluorescence, using the Agilent Small RNA kit on the Agilent Bioanalyzer 2100, and the Qubit^® microRNA assay (Life Technologies, Gent, Belgium) on the Qubit^® 2.0 Fluorometer. For Agilent Small RNA assay, 1 μL of RNA from the stock solution or from equally diluted samples was used. For Qubit^® microRNA assay, 10 μL of equally diluted RNA samples were used. Electropherograms were visualized using the Agilent 2100 Expert software (Agilent technologies).

For RNA isolation, each bone matrix was transferred from -80°C storage to liquid nitrogen in a pre-chilled mortar on ice and quickly homogenized with 10 volumes of Lysis/binding buffer, avoiding any partial thawing. Homogenized samples were transferred to pre-chilled tubes and both total RNA and miRNA were extracted using the mirVana^™ miRNA Isolation Kit (Life Technologies) following manufacture’s recommendations. RNA quality and yield were first determined by NanoDrop spectophotometer and then by Agilent 2100 Bioanalyzer using Agilent RNA 6000 Nano and Agilent small-RNA Lab-Chip kits. All samples presenting RNA integrity number (RIN) equal or higher than 8 were considered suitable for the downstream applications.
Extract labelling and hybridization on chips were performed according to standard protocols compliant with Affymetrix technology.

Total RNA from PBMCs was extracted using TRIzol (Invitrogen). The integrity of the RNA was measured with Agilent RNA 6000 Nano. SuperScript™ III One-Step RT-PCR(Invitrogen) was used for reverse transcription and amplification. Quantitative PCR of CSRnc transcripts for IGHM, IGHG1, IGHG3, and AID gene was performed using specific primers and TaqMan probes(IDT). The primers and probes used to quantify the CSRnc transcripts are detailed in Table 1. Amplification of HPRT with PrimeTime® Predesigned qPCR Assays was performed as the reference gene. The fold difference was calculated using 2^ΔΔCT, using resting enriched B cells as calibrator and non-B cells as negative control. HPRT was used as normalizer for every condition.

MiRNAs were isolated using the miRNA kit (Qiagen, Hilden, Germany) according to the manufacturer instructions. The quantity and quality of RNA were assessed using the NanoDrop ND-1000 instrument (Nanodrop Technologies, Wilmington, USA) and the Bioanalyser 2100 system (Agilent Technologies, CA, USA) using the Agilent RNA 6000 Nano according to the manufacturer instructions. MiRNAs were profiled by NGS on the Illumina Hiseq 2000 instrument (Illumina Inc., San Diego, USA) using the Illumina TruSeq Rapid SBS preparation protocol (Arraystar Inc., Rockville, USA). The cDNA library was prepared using the SMART cDNA Library Construction Kit (Clontech Laboratories, Inc. CA, USA) according to the manufacturer instructions. The quantification of the cDNA library was measured using a 2100 Bioanalyser (Agilent Technologies), with the final concentration being 10 pM. Differentially expressed miRNAs between the two groups were analyzed using the two-tailed, homoscedastic t-test.

The SH-SY5Y cells were cultured in 75 cm² tissue culture plates using standard media conditions. Upon reaching 90% confluency, media was aspirated, and cells were washed once with ice-cold PBS and then harvested with TrypLE (Gibco, Waltham, MA, USA). Following collection, cells were pelleted by centrifugation 1500× g, for 5 min, at 4 °C. After pelleting, cells were washed 1× in ice-cold PBS and again pelleted by centrifugation 1500× g, for 5 min, at 4 °C. Following pelleting, RNA was isolated using TRIzol reagent (Ambion, Austin, TX, USA). Here, 1 mL of TRIzol was added to cell pellets, triturated, and RNA extracted according to the manufacturer’s suggested protocol including isopropanol precipitation and 70% EtOH wash. Following resuspension of the RNA, RIN was assessed by Agilent 2100 Bioanalyzer with the Agilent RNA 6000 Nano before submission for sequencing at the Beijing Genomics Institute (BGI). Two independently thawed, passaged, and harvested cell populations were used for transcriptomic analysis. Here, RNA sequencing was conducted for two biological replicates.