Nextflex rapid directional qrna seq kit

Total RNA was extracted from the bulk sorted microglia samples using the RNeasy Plus Micro kit (Qiagen, 74034) according to the manufacturer’s protocol. RNA quality and quantity were determined with the Experion RNA HighSens Analysis Kit (Bio-Rad, #7007105). All 25 RNA samples, with RIN values varying between 5.1 and 9.9, were enriched for poly(A) + messenger RNA using NEXTflex Poly(A) Beads (BIOO Scientific, #NOVA-512980) according to the manufacturer’s protocol, and 14 μl of this mRNA-enriched poly(A)-tailed RNA was used as the input for the NEXTflex Rapid Directional qRNA-Seq kit (BIOO Scientific, #NOVA-5130-04). Library preparation was performed according to the manufacturer’s protocol. Quality and concentration of libraries from individual samples were assessed using the High Sensitivity dsDNA kit (Agilent, 067-4626) on a 2100 Bioanalyzer (Agilent) and a Qubit 2.0 Fluorometer (Life Technologies). Subsequently, individual libraries were combined into two sequencing pools of 13 samples each with equal molar input, and 75 bp paired-end sequencing was performed on an Illumina NextSeq 500 system. PhiX was added at 5% to both pools as an internal control before sequencing.

BMDMs were treated with GABA for the indicated times and washed with PBS. Total RNA was extracted from each sample using TRIzol (Invitrogen). An RNA sequencing library was generated using Nextflex Rapid Directional qRNA-Seq Kit, in accordance with the user’s instruction manual (Bioo Scientific, Austin, TX, USA). Briefly, mRNA was purified from total RNA using Oligo (dT) beads and was chemically fragmented. After double-strand complementary DNA (cDNA) synthesis of the fragmented mRNA, adenylation of the 3′-end, sequencing adapter ligation, Uracil-DNA Glycosylase (UDG) treatment, and PCR amplification were performed, followed by DNA purification with magnetic beads. Finally, the amplified library was checked with BioAnalyzer 2100 (Agilent, CA, USA) and then applied for sequencing template preparation. The HiSeq2500 platform was utilized to generate 100-bp paired-end sequencing reads (Illumina, CA, USA).

After isolation of total RNA the RNA integrity was analyzed with the RNA 6000 Nano Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies). From total RNA, mRNA was extracted using the NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs) and RNA-Seq libraries were generated using the NEXTFLEX Rapid Directional qRNA-Seq Kit (Bioo Scientific) as per the manufacturer’s recommendations. Concentrations of all samples were measured with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and fragment lengths distribution of the final libraries was analyzed with the DNA High Sensitivity Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies). All samples were normalized to 2 nM and pooled equimolar. The library pool was sequenced on the NextSeq500 (Illumina) with 1 × 75 bp, with 16.1 to 18.6 mio reads per sample.
For each sample the sufficient quality of the raw reads was confirmed by FastQC v0.11.8 [54 ]. Afterwards, the reads were aligned to the mouse reference genome GRCm38 with STAR v2.6.1c [16 (link)] and simultaneously counted per gene by employing the --quantmode GeneCounts option. Counts are based on the Ensembl annotation release 95. Differential expressed genes were estimated with DESeq2 v1.22.2 [37 (link)].

Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturers’ protocol from 3 samples from each group collected 7 days post-amputation. mRNA was purified with NEXTflex Poly(A) beads (Bioo Scientific). RNA libraries were prepared with NEXTflex® Rapid Directional qRNA-Seq™ Kit (Bioo Scientific) following the manufacturer’s protocol and sequenced on an Illumina HiSeq2500 machine.

Total RNA was extracted from whole cells using TRIzol Reagent following the manufacturer’s instructions. Isolated RNA was subjected to ribosomal RNA depletion using the Ribo-Zero rRNA Removal Kit (Illumina). RNA-seq libraries were prepared using the NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific) following the manufacturer’s instructions and sequenced using 75 bp single-end sequencing on the Illumina NextSeq 500 platform by the BioFrontiers Institute Next-Gen Sequencing Core Facility.