Project description:Many library preparation methods are available for gene expression quantification. Here, we sequenced and analysed Universal Human Reference RNA (UHRR) prepared using Smart-Seq2, TruSeq (public data) and a protocol using unique molecular identifiers (UMIs) that all include the ERCC spike-in mRNAs to investigate the effects of amplification bias on expression quantification. UHRR 10 and 12 replicates for Smart-seq2 and UMI-seq library preparation methods, respectively.
Project description:Many library preparation methods are available for gene expression quantification. Here, we sequenced and analysed Universal Human Reference RNA (UHRR) prepared using Smart-Seq2, TruSeq (public data) and a protocol using unique molecular identifiers (UMIs) that all include the ERCC spike-in mRNAs to investigate the effects of amplification bias on expression quantification.
Project description:We assessed Smart-Seq, a new single-cell RNA-Seq library preparation method, on a variety of mouse and human RNA samples or cells. We generated RNA-Seq libraries for dilution series of MAQC reference RNA and mouse brain RNA to assess technical reproducibility, and for a variety of individual cells including putative circulating tumour cells.
Project description:Purpose: The aim of this study is to compare different long-read sequencing platforms using reference lung adenocarcinoma cell lines and spike-in controls. Methods - Cell Culture: Lung adenocarcinoma cell lines NCI-H1975 and HCC827 from a range of passages (2-4) were grown on 3 separate occasions in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum and 1% penicillin-streptomycin. Methods - RNA preparation: mRNA was extracted using a Qiagen RNA miniprep kit and purified using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490). Purified mRNA spiked with sequins was used for Next Generation Sequencing library preparation using the NEBNext Ultra II Directional RNA Library Prep Kit (Illumina) and the cRNA-PCR Barcoding (SQK-PCS109 with SQK-PBK004) kit (ONT). Completed libraries were sequenced on NextSeq 500 (Illumina) and PromethION (ONT). Iso-Seq libraries were prepared and sequenced by Novogene on Sequel II (PacBio). Reads were mapped to known genomic features of the GRCH38 reference genome and RNA sequin decoy chromosome combined sequences at the gene-level and single reads were then summarized into gene-level counts using featureCounts software (Liao et al. 2014).
Project description:Here, we report the development of two RNA-seq library preparation protocols that increase the throughput and decrease the cost of converting RNA to cDNA libraries compatible for sequencing on high-throughput platforms. BaM-seq allows for early barcoding of samples such that many biological samples can be processed simultaneously. TBaM-seq allows for enrichment of target RNAs to decrease the required sequencing depth. Both methods are able to accurately measure gene expression changes with high technical reproducibility and agreement with gold standard, lower throughput approaches.
Project description:Here, we report the development of two RNA-seq library preparation protocols that increase the throughput and decrease the cost of converting RNA to cDNA libraries compatible for sequencing on high-throughput platforms. BaM-seq allows for early barcoding of samples such that many biological samples can be processed simultaneously. TBaM-seq allows for enrichment of target RNAs to decrease the required sequencing depth. Both methods are able to accurately measure gene expression changes with high technical reproducibility and agreement with gold standard, lower throughput approaches.
Project description:We report novel single-cell RNA-Seq, called Quartz-Seq. Quartz-Seq was simplified method compared with previous methods based on poly-A tailing reaction. RNA-seq by illumina TruSeq, KAPA library preparation kit, single-cell Quartz-Seq and single-cell Smart-Seq by illumina HiSeq 2000/1000
Project description:We have developed two methods for efficiently consructing RNA-seq libraries using transposition. Each method constructs high quality RNA-seq libraries when compared to standard approaches. One of the methods (Directional Tn-RNA-seq) maintains strand-of-origin information and exhibits strand specificity comparable to current approaches. RNA-seq libraries were constructed from ECC-1, a human cell line, and Universal Human Reference RNA using transposon-based and standard RNA-seq library construciton methods.
