Project description:ATAC-seq of Col and hcr2 mutant from seedling and bud sample. Tagmentation was performed using the Tag DNA enzyme & buffer kit (Illumina, 20034210). Nextera DNA CD Index primers were used for library construction. The indexed libraries were subjected to paired-end 50-bp sequencing using an Illumina HiSeqX instrument (Microgen, Korea).
Project description:Purpose: We used GAS as a model bacterial pathogen to investigate the complex relationship between genome, transcriptome, and virulence in a large population of type emm28 strains recovered from humans over 26 years in geographically diverse locations. Methods: We chose a subset of isolates from the sequenced population of 2,101 emm28 strains that would approximately span the genetic variation present in the entire population. Fifty emm28 strains were grown in triplicate, harvested at two time points, mid-exponential (ME) and early-stationary (ES) phases of growth, and assayed in triplicate by RNA-seq. rRNA was depleted using the Ribo-Zero rRNA removal kit for Gram-positive bacteria (Illumina) and libraries were made using the ScriptSeq complete kit for bacteria (Illumina). The cDNA libraries were prepared with indexed reverse primers from the ScriptSeq Index PCR primers kit (Illumina), and purified with AMPureXP beads (Beckman Coulter).
Project description:For analysis of mRNA expression levels, total RNA was harvested from each cell-line in replicate with Trizol™ (Thermo scientific). Total RNA was purified using Direct-zol™ columns according to the manufacturers specifications (Zymo Research). For cDNA synthesis 1 μg of total RNA was process as the T12VN-PAT assay (Jänicke et al., RNA 2012), except that this was adapted for multiplexing on the Illumina MiSeq instrument. We refer to this assay as mPAT for multiplexed PAT. The approach is based on a nested-PCR that sequentially incorporates the Illumina platform’s flow-cell specific terminal extensions onto 3’ RACE PCR amplicons. First, cDNA was generated using the anchor primer mPAT Reverse, next this primer and a pool of 50 gene-specific primers were used in 5 cycles of amplification. Each gene-specific primer had a universal 5’ extension (see supplementary file primers) for sequential addition of the 5’ (P5) Illumina elements. These amplicons were then purified using NucleoSpin columns (Macherey-Nagel), and entered into second round amplification using the universal Illumina Rd1 sequencing Primer and TruSeq indexed reverse primers from Illumina. Second round amplification was for 14 cycles. Note, that each experimental condition was amplified separately in the first round with identical primers. In the second round, a different indexing primer was used for each experimental condition. All PCR reactions were pooled and run using the MiSeq Reagent Kit v2 with 300 cycles (i.e. 300 bases of sequencing) according to the manufacturers specifications. Data were analysed using established bioinformatics pipelines (Harrison et al., RNA 2015)
Project description:sRNAs were cloned and sequenced from the wild type Arabidopsis fwa epimutant, and the Arabidopsis dcl2/4 mutant harbouring the fwa epimutant. Both wild type and dcl2/4 were infected with mock conditions or modified Tobacco Rattle Virus (TRV) containing a portion of the FWA promoter sequence (TRV:FWAtr). Libraries were indexed during PCR amplification (12 cycles) according to the Illumina protocol. See individual sample information for specific index primers used.
Project description:RNA was extracted from mice and mRNA isolated from 2ug total RNA. mRNA was processed to generate a library compatible with Illumina high throughput sequencing using a standard RNA-seq library generation protocol. Each library was amplified using indexed primers and samples were purified using AMPure XP beads. Libraries were pooled and quantified with Bioanalyzer and qPCR.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:Two tree-man syndrome lesions immersed in RNA later were snap-frozen in liquid nitrogen and crushed with a tissue pulverizer kit (Cell crusher) according to the manufacturer’s instructions. Total RNA was extracted with the RNeasy Fibrous Tissue Mini Kit (QIAGEN), according to the manufacturer’s instructions. The procedure included a DNAse digestion step or an additional column to remove contaminating DNA. The concentration and purity of the total RNA extracted were measured by spectrometry with an Xpose (Trinean). RNA integrity was evaluated by capillary electrophoresis with a Tape Station (Agilent). The Ovation Universal RNA-Seq System from NUGEN was used to prepare the RNA-seq libraries from100 ng of total RNA, as recommended by the manufacturer. This type of RNAseq kit is less sensitive to total RNA degradation (the RNA integrity number (RIN) of the two samples was quite low: 3.9 for one sample and 5 for the other). This kit constructs strand-specific RNA-seq libraries from 10 to 100 ng of total RNA and uses Insert-Dependent Adaptor Cleavage(InDA-C) technology to remove the ribosomal RNA transcripts. The RNA is subjected to reverse transcription and the second strand is synthesized and a fragmentation step is then performed before Illumina-compatible indexed adaptator ligation. This ligation is followed by a strand-selection enzymatic reaction to provide information about the sense of the transcripts. Insert-dependent adaptor cleavage (InDA-C)-specific primers were then used for the targeted depletion of human ribosomal RNA sequencing transcripts before PCR enrichment. We ensure that no excess amplification occurred during the final PCR step, by evaluating the number of PCR cycles to be applied to each sample in a preliminary Q-PCR test with EvaGreen. An equimolar pool of the final indexed RNA-Seq libraries was sequenced on an Illumina NovaSeq6000 (paired-end reads, 100 bases + 100 bases) and ~50 million paired-end reads per library were produced.