Project description:We compared 3 small RNA library prep kits (CleanTag, NEXTflex, QIAseq) and two RNA extraction methods (miRNeasy and MagnaZol) on plasma. We report that library preparation has a significant effect upon the miRNA profile detected, with QIAseq libraries exhibiting the least sequencing bias of the three library kits. RNA extraction methods also contribute, to a lesser extent, to the miRNA profile detected, with MagnaZol RNA extraction increasing the percentage of reads mapping to miRNAs and the number of individual miRNAs detected.
Project description:Purpose: The study aimed to characterize the molecular phenotype of bone marrow macrophages in NHL with RNA sequencing analysis. Methods:RNA of sorted femur macrophages (CD11b+F4/80+) were extracted with an RNA extraction kit (Qiagen). The samples were submitted to Novogene Inc. for library preparation and subsequent RNA sequencing. RNA was used for cDNA library construction using an NEBNext® Ultra RNA Library Prep Kit for Illumina® (New England Biolabs) according to the manufacturer’s protocol. The resulting 250-350 bp insert libraries were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and quantitative PCR. Size distribution was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Qualified libraries were sequenced on an Illumina HiSeq 4000 Platform (Illumina) using a paired-end 150 run (2×150 bases). Reads containing adapter or poly-N and those of low quality were trimmed, after which gene counts were obtained though mapping the clean reads to reference genome mm10 using STAR 2.5.3a.
Project description:This study provides ultra-deep bulk RNA-seq data from human embryonic stem cell (hESC)-derived thyroid follicular cells. The NKX2-1GFP+ population was isolated using fluorescence-activated cell sorting (FACS) and subjected to RNA extraction. Library preparation was performed using the Ovation Solo RNA-seq System, followed by high-output sequencing on the Illumina HiSeq 1500 platform. Each sample generated approximately 400 million reads. These data enable transcriptomic analysis of differentiated thyroid lineage cells exposed to IFN-α and IFN-γ.
Project description:The murine lung tissues from Vehicle (V) group, LPS (L) group, and LPS+TSLP (T) group were collected to perform next-generation sequencing (NGS). Illumina RNA Library Prep kit was used to perform library preparation and then paired-end library sequencing was performed by Illumina NovaSeq 6000 and sequence analysis was determined using the Illumina data analysis pipeline.
Project description:Synchronised animals were grown to young adult stage at 20C on HB101 seeded NGM plates. Animals were harvested and washed 3X in M9 buffer. Settled animals were mixed with Trisure reagent, bead beaten using zirconia beads, and the RNA is extracted using chloroform. For total RNA sequencing, Illumina Ribozero kit is used to remove ribosomal RNA from 1ug of total RNA prior to library preparation. RNA sequencing libraries are prepared using NEB Next Ultra library preparation kit. Small RNA sequencing is performed by treating 5ug of total RNA with Epicentre 5 polyphosphatase to remove the 5 triphosphate. After treatment, RNA is purified by phenol/chloroform extraction and 1ug of RNA is used to prepare small RNA libraries using Illumina TruSeq small RNA library preparation kit. Ribosomal depleted RNA and small RNA libraries are sequenced using Illumina HiSeq 1500 platform.
Project description:We dissected foreguts from Gli2-/-,Gli3-/- and Gli2+/-,Gli3+/- mouse embryos at embryonic day 9.5. Three foreguts were obtained for each group and each foregut was individually processed for bulk RNA extraction and library preparation. Sequencing results identified Gli positively and negatively regulated genes in the foreguts.
Project description:The goal of the study was to compare gene expression of Robo1+/+ and Robo1-/- luminal progenitors. Total RNAs were then extracted from FACS purified luminal progenitor cells, harvested from Robo1+/+ or Robo1-/- mice (n=3 per genotype, two animals per n) using TRIreagent LS (Sigma, T3934). Poly(A)+ RNA sequencing libraries were made from each sample using the TruSeq RNA library preparation kit v.1 (Illumina). Illumina RNA PolyA library preparation guide. A total of 6 libraries were created by PCR amplification with Illumina barcoding primers using kit recommended conditions and quantified using a Bioanalyzer DNA 1000 kit (Agilent).
Project description:We evaluated the effect of the small RNA library preparation method on 5' tRNA-halves and miRNA abundance in libraries prepared from serum RNA using three commercially available small RNA library preparation kits (TruSeq small RNA library preparation kit v2 (Illumina), TailorMix miRNA sample preparation kit v2 (Seqmatic) and the NEBNext Multiplex Small RNA library prep kit (New England Biolabs)). RNA isolated from 100 µl of serum collected from healthy mice was used as input for the preparation of a small RNA library in duplicate and libraries were single end sequenced.