Truseq rna sample preparation kit

Four purified RNA extracts (2 from cells grown in glucose supplemented medium and another 2 from cells grown in PE supplemented medium) with the best RIN values were selected for sequencing. First, ribosomal depletion was performed using Ribo-Zero Magnetic Kit (Epicentre, USA). Then cDNAs were synthesized using TruSeq RNA Sample Preparation Kit (Illumina, USA) and SuperScript II Reverse Transcriptase (Invitrogen, USA). A minimum of 20 ng cDNA was fragmented using Covaris S220 (Covaris Inc., USA) to a targeted size of <500 bp. The fragmented cDNA were then end-repaired, ligated to Illumina TruSeq Adapters and PCR-enriched using TruSeq RNA Sample Preparation Kit (Illumina, USA). The final sequencing libraries were quantified using KAPA kit (KAPA Biosystem, USA) on a Stratagene Mx-3005P qPCR system (Agilent Technologies, USA). Library sizes were confirmed using Agilent Bioanalyzer High Sensitivity DNA Chip (Agilent Technologies, USA). The resulting libraries were subjected to cluster generation and sequenced using an Illumina flow cell, 202 cycles (101 bp paired-end reads) on the Illumina HiSeq 2000 system (Illumina, USA). All steps were performed according to the manufacturers’ protocol, unless otherwise stated.

In general, the larval midgut is the principal place to detoxify the exogenous 20E. Therefore, the RNA, which was extracted from the midgut collected at 3 h after 20 μg 20E treatment, was subjected to transcriptomic analysis. Each midgut sample was collected from five larvae and ground in liquid nitrogen to a powder. Total RNA was extracted using a TransZoL up Plus RNA Kit according to the manufacturer’s protocol (Beijing TransGen Biotech, Beijing, China). RNA purity was checked using a Nano Photometer spectrophotometer (Implen, Westlake Village, CA, USA). RNA 6000 Nano Assay Kit and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) were implemented to assess RNA integrity. Then, 3 μg of total RNA per sample was used as input material for RNA sequencing. The transcriptome libraries were generated using Illumina TruSeq™ RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer’s recommendations. RNA-Seq based transcriptome profiling was performed by Biomarker Technologies Corporation (Beijing, China). NovaSeq 6000 platform was applied to generate 150 bp paired-end reads. Each biological sample was repeated two times. The raw data of RNA-seq has been deposited into China National Center for Bioinformation (ID: CRA006208).

Total RNA was extracted from developing seeds using TRIzol Reagent (Invitrogen, Carlsbad, CA). RNA pellets were dissolved in RNAse-free water, quantified by NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). RNA quality was checked by 2% agarose gel electrophoresis. Total RNA from the 30 DAP sample was used for preparing an mRNA sample and subsequently constructing of a cDNA library using Illumina ® TruSeq™ RNA Sample Preparation Kit (Illumina Inc., San Diego, CA ). In brief, the mRNA was purified using poly-T oligo attached to magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then went through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final cDNA library. The cDNA library was sequenced on a GS FLX Titanium sequencing platform (Roche, Branford, CT).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer instructions. RNA integrity was determined using the RNA6000 Nano kit on the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). RNA libraries were prepared using the low-throughput protocol of the Illumina TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA). Briefly, 1 μg of total RNA was used for poly-A mRNA selection, and selected mRNA was then fragmented in high-salt and high-temperature conditions. cDNA synthesis, end-repair, A-tailing, multiplex indexing, and PCR amplification was performed and the resulting libraries were quantified and analyzed on a HiSeq2000 (Illumina) for SR50. The resulting reads were aligned to the Hg19 transcriptome. Data analysis was completed using DESeq version 1.6.159 (link).

CH and CL flower libraries were generated from flower buds, and each flower bud sample consisted of a mixture of flower buds from different developmental stages obtained from 50 individuals of the same P. heterophylla population. For each kind of flower, total RNA was isolated from the flower buds using Trizol Reagent (Invitrogen) and treated with DNaseI (Promega) to remove residual contaminating DNA. The quality and integrity of the RNA were determined using an Agilent 2100 Bioanalyzer (Agilent). Two RNA-seq libraries (e.g. CH flowers and CL flowers) were prepared using the Illumina TruSeqRNA Sample Preparation Kit (Illumina) following the manufacturer’s instructions. Two normalized cDNA libraries (CH flowers, CL flowers) were constructed and sequenced using the Illumina HiSeq platform (Shanghai Personal Biotechnology Co. Ltd, China) to generate 100 bp paired-end raw reads.

Samples of A. craccivora (about 300 heads) were collected from locust trees on May 2015 in Zhengzhou of China (the geospatial coordinates: 34.723°N, 113.635°E). Total RNA was extracted from 35 to 50 winged adult individuals by Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s procedure. The total RNA quantity and purity were determined using a Bioanalyzer 2100. Sequencing libraries were constructed using IlluminaTruSeq™ RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). The results of RNA quantity and purity checking were the following: the concentration was around 3.15 ug/ul, A260/280 was 2.09, A260/230 was 1.52, total content was 300 ug, 28S/18S was 1.1, and RIN was 8.0. Prior to sequencing, two samples were prepared for repeat checking. The best run was used for further analysis. The RNA transcript was sequenced on an Illumina (Solexa) GAII sequencing machine in Shanghai OE Biotech CO., LTD. The sequencing depth was set to 4 Gb Raw data for each sample.