Project description:HT29-DKO cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, 300 million HT29-DKO cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the Brunello library at MOI 0.3. Cells were then selected with puromycin, expanded to 3 billion cells, and then pooled together and cryofrozen in aliquots. One hundred million cells were thawed constituting over 1000× genome coverage worth of mutagenized library. The cells were infecting with PeV-A1 or PeV-A2 at an MOI of 0.1. Virus-resistant colonies were harvested. The uninfected reference used was the unselected starting population. The unselected and selected cells were both processed with QIAamp DNA columns to purify the gDNA. A first round of PCR was used to amplify the guide RNA sequences encoded in the gDNA, followed by a second round of PCR to add the barcodes/adapters for amplicon sequencing. 2% agarose gels and a QIAquick gel extraction kit were used to purify the amplicons. The amplicons were then subjected to next-generation sequencing on a HiSeq instrument lane (Illumina) via Novogene.
Project description:The aim of the experiment was to assay every gene in the E. coli genome to identify those that contribute to increased or decreased susceptibility to the beta-lactam antibiotic meropenem. A library of transposon-insertion mutants was grown overnight in the presence or absence of a range of concentrations of meropenem. Different concentrations of IPTG were also used to promote expression from a transposon-encoded promoter to assay the effects of increased transcription for each gene. DNA sequencing was then used to reveal the locations of the transposons in every mutant. By comparing the numbers of each mutant between conditions, information can be gained about the relative fitness of that mutant under the conditions tested.
Project description:We applied Solexa sequencing technology to identify Singapore grouper iridovirus (SGIV) encoded microRNAs during its infection. A small RNA library arising from SGIV infected grouper cells (GP) was constructed and sequenced. We recovered 6,802,977 usable reads, of which 34,400 reads represented the small RNA sequences encoded by SGIV. Among them, 16 novel SGIV encoded miRNAs were identified by a computational pipeline. Generally, these 16 miRNAs are dispersed throughout the SGIV genome, while three of them are located within open reading frame 057L (ORF057L) region. Meanwhile, We identified 138 conserved microRNA genes between grouper fish and zebrafish.
Project description:YAV20 (E7946 loxP[dciA] SpR lacZ::cre ZeoR), AB14 (YAV20 inv[glmU-mioC]) and AB23 ( YAV20 ΔrecB::CmR) were created by natural transfromation using cognate plasmids. Cells were grown in the M( minimla media supplemented with fructose, with or without arabinose (ara). Genomic DNA was extracted with the Sigma GenElute® bacterial genomic DNA kit to generate a genomic library according to Illumina’s protocol. The libraries and the sequencing were performed by the High-throughput Sequencing facility of the I2BC (https://www.i2bc.paris-saclay.fr/sequencing/ng-sequencing/, CNRS, Gif-sur-Yvette, France). Genomic DNA libraries were made with the ‘Nextera DNA library preparation kit’ (Illumina) following the manufacturer’s recommendations.
Project description:High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. Application of this method allows whole genome studies from samples where material or yields are limiting. Comparison of ChIP-seq libraries constructed from 100 pg DNA (this study) and nanograms of DNA (modENCODE). ChIP antibody: H3K27me3, Active Motif 31955.
Project description:Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (1) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (2) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (3) achieved paired-end sequencing of the ChIP-seq libraries; (4) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq; and (5) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies. Pre-adipocytes and mature adipocytes were collected. Their chromatin and RNA were subjected to ChIP and mRNA extraction. Sequencing libraries from ChIP DNA or mRNA were generated following either standard protocols or TELP method. The quality and features of TELP libraries were proved and demonstrated in comparison with standard libraries or other published data.
Project description:Whole genome sequencing of 10 HCLc tumor and matched-germline T cells. Genomic DNA from highly purified HCLc tumor and T cell populations were utilized for library preparation using NEBNext Ultra DNA library prep kit. Sequencing was performed as 150 bp paired end sequencing using four lanes of an Illumina HiSeq4000 to an average depth of 12X. Reads from each library were aligned to the human reference genome GRCh37 using BWA-MEM (v0.7.12). The analysis of somatic genetic alterations in WGS data from tumor-germline pair HCLc samples was divided based on the nature of the mutation, as follow: single-nucleotide variants (SNVs), indels, CNAs and SVs. Moreover, COSMIC mutational signatures and subclonal architecture was inferred for each tumor.
Project description:eHAP or U87MG cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, 300 million eHAP or U87MG cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the Brunello library at MOI 0.3. Cells were then selected with puromycin, expanded to 3 billion cells, and then pooled together and cryofrozen in aliquots. One hundred million cells were thawed constituting over 1000× genome coverage worth of mutagenized library. Twenty T175 flasks were used for the U87MG-based screen, while twelve T175 flasks were used for the eHAP-based screen. The cells were allowed to recover for 48 hours before infecting with ReoT3D at an MOI of 0.1. Obvious CPE was observed within 72 hours. eHAP-resistant colonies were harvested two weeks later, while U87MG-resistant colonies were harvested six weeks later. The uninfected reference used was the unselected starting population. The unselected and selected cells were both processed with QIAamp DNA columns to purify the gDNA. A first round of PCR was used to amplify the guide RNA sequences encoded in the gDNA, followed by a second round of PCR to add the barcodes/adapters for amplicon sequencing. 2% agarose gels and a QIAquick gel extraction kit were used to purify the amplicons. The amplicons were then subjected to next-generation sequencing on a HiSeq instrument lane (Illumina) via Novogene.