Quantseq 3 mrna seq kit

RNA-seq libraries for the density fractionation were generated using 1 μg of RNA as starting material and depleting ribosomal RNA using Ribocop V3 (Lexogen). Post ribosomal RNA depletion, RNA content was measured with a Bioanalyzer pico kit (Agilent). Then 1% RNA spike-in RNA variants (SIRVs) were spiked-in (Lexogen), and RNA-seq libraries were generated and amplified using the CORALL RNA-seq kit (Lexogen) according to the manufacturer’s instructions. All CORALL-generated libraries were sequenced in parallel on four Novaseq S4 lanes (Illumina). RNA-seq libraries for the differential sedimentation speed based cell fractionation experiment were performed using QuantSeq 3′ mRNA-Seq kit (Lexogen) according to manufacturer’s instructions. A total of 400 ng of total RNA and 0.1% RNA SIRVs (Lexogen) were used for library preparation of each fraction. All libraries were balanced, multiplexed and pooled, and run on two single-end Novaseq SP lanes (Illumina).

RNA sequencing was performed at the RNA-Seq Center at the Division of Pulmonary and Critical Care, Feinberg School of Medicine, NU. Following RNA extraction, the RNA integrity number (RIN) was measured using the Agilent TapeStation 4200 (Additional file 1: Figure S1A and S1B). RNA-seq libraries were prepared using a QuantSeq 3′ mRNA-seq kit (Lexogen). Fragment size distribution for the libraries was assessed using the TapeStation 4200. Libraries were multiplexed and sequenced on the NextSeq 500 platform (Illumina) to an average depth of 7 × 10⁶ single-end reads. FASTQ files were processed using the QuantSeq 3′ mRNA-seq pipeline implemented on the Bluebee genomic platform (Bluebee) with the following steps: files were trimmed with BBDuk and aligned with STAR to the human genome (GRCh38.77), and a table of gene counts was generated from aligned reads with HTSeq. A MultiQC report [19 (link)] was created to evaluate RNA sequence quality (non-normalized data are shown in Additional file 2: Table S1, and normalized counts are shown in Additional file 3: Table S2, S3 and S4, and Additional file 4: Table S5).

To extract cellular RNA, entire tissues were immersed in Trizol and lysed by bead beating for five minutes at a 20 s⁻¹ frequency using the Qiagen Tissue Lyzer with the Navy bead beating kit (Next Advance). Tissues homogenates were further lysed by passing through the Qiashredder (Qiagen). Homogenates were then extracted with chloroform, mixed with an equal volume of ethanol, and loaded into columns of an RNeasy mini kit (Qiagen), at which point the manufacturer’s directions were followed, which included the on-column DNAse (Qiagen) digestion. RNA concentrations were determined using a Quant-it RNA Assay Kit with a Qubit 3.0 Fluorometer. Library preparation was performed using the QuantSeq 3′ mRNA-Seq kit (Lexogen), which using oligo-dT priming and does not require the use of poly(A) enrichment or rRNA depletion [45 ]. Libraries were sequenced on a NextSeq using the 500/550 High Output v2 kit (Illumina) in 75 base pair single-read mode.