Scalable full-transcript coverage single cell RNA sequencing of PBMCs using Smart-seq3xpress
Ontology highlight
ABSTRACT:
INSTRUMENT(S): DNBSEQ-G400
ORGANISM(S): Homo sapiens
SUBMITTER: Christoph Ziegenhain
PROVIDER: E-MTAB-11452 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Smart-seq3xpress was carefully optimized and >1,000 conditions were evaluated. This data submission is organized in 15 datasets that each contain fastq files, unmapped bam files, read count tables, UMI count tables and a barcode annotation file. The barcode_annotation.txt files contain the exact factors/variables tested. Below a short description of each set of experiments: K562_lowvolume: Evaluation of scaling volumes of Smart-seq3 (indicated volume refers to total volume in PCR), whether overlay was used and if cDNA was bead-cleaned or diluted prior to tagmentation. Cell input was K562 cells. The columns \\"treatment\\", \\"volume\\" and \\"VL\\" indicate the experimental parameters. HEK_lowvolume: Evaluation of scaling volumes of Smart-seq3 (indicated volume refers to total volume in PCR), whether overlay was used and if cDNA was bead-cleaned or diluted prior to tagmentation. Cell input was HEK293FT cells. The columns \\"treatment\\", \\"volume\\" and \\"VL\\" indicate the experimental parameters. overlays: Evaluation of the effect of various overlays when generating HEK293FT libraries in 1 uL total volume. The column \\"condition\\" indicates the applied overlay. tagmentations: Evaluation of input cDNA vs Tn5 amount during tagmentation. Purified cDNA from one 384-well plate was used as input into various conditions of tagmentations. The experiments contain evaluation of cDNA amount with fixed Tn5 amount or varying Tn5 amount while keeping the default volume (2 uL) of the tagmentation reaction or scaling the reaction volume. The column \\"condition\\" contains a string indicating reaction volume, cDNA input and Tn5 ATM enzyme amount. If no volume is indicated, reaction was performed in 2 uL. HomeTn5: Evaluation of tagmentation using in-house produced Tn5 enzyme (Picelli et al., 2015) when tagmenting cDNA generated from HEK293FT cells in 1 uL total volume. The column \\"Tn5concentration\\" indicates the Tn5 reaction concentration at 2 uL reaction volume. cycles_cleanups: Optimization of Smart-seq3xpress (column \\"experiment\\" shows \\"direct_tag\\") in regards to clean-ups after cDNA synthesis (column \\"condition\\": noclean, Exo+FastAP, ExoSAP) and dilution volume (9 or 19 uL); PCR cycle numbers (column \\"pcr_input\\") and ATM Tn5 enzyme amount (column \\"ATM\\"). Cell input was HEK293FT cells. PreAmp_Polymerase: Evaluation of various PCR polymerases during initial cDNA amplification. The polymerases are indicated in column \\"polymerase\\". We also evaluated several TSO concentrations (concentration in RT is given) and fwd/rev PCR primers (concentration given in PCR reaction). Cell input was HEK293FT cells. TDE1: Large optimization of tagmentation conditions using the TDE1 Tn5 enzyme. We varied reactions by changing PCR polymerase (KAPA / SeqAmp), PCR extension time and the number of PCR cycles during cDNA amplification. During tagmentation, we varied the amount of TDE1 enzyme, the amount of DMF in the tagmentation reaction buffer and the presence of Tween-20 in the final post-tagmentation PCR. Cell input was HEK293FT cells. TSOs_RT_v1-7: Large scale evaluation of conditions relating to RT and PCR, with a focus on new template-switching oligo (TSO) designs. In total, >20,000 cells and >500 conditions are contained in these datasets. The barcode annotation file contains precise information on the reaction conditions of Lysis, RT, PCR as well as utilized TSO designs. Data was generated from HEK293FT cells and hPBMC (Lonza).
2022-05-16 | E-MTAB-11467 | biostudies-arrayexpress
Project description:Molecular characterization of perinatal liver HSCs.
2023-09-30 | E-MTAB-13293 | biostudies-arrayexpress
Project description:Individual HEK cells were dispensed using an F.SIGHT into individual wells while recording cell diameters. Each well contained 0.0321 pg of molecular spike-ins, a highly complex set of 264 molecular spikes, based on 11 unique spike sequences spanning different lengths (570 to 3070 nts) and GC contents (40-60%). Libraries were generated with Smart-seq3xpress protocol.
2022-02-16 | E-MTAB-11433 | biostudies-arrayexpress
Project description:To study cancer cells heterogeneity at the single cell level we grew cancer cells as spheroids and extracted their RNA preform SmartSeq3xpress. We grew MDA-MB-231 cells on agar coated plates for 5-10 days in DMEM 10% FBS. The spheroids were incubated for 2 hours with Calcein AM and Vybrant Dye 10uM at 37C and washed twice with PBS. After dissociation with trypsinLE 0.25% the cells were facs sorted and the fluorescence intensity for each cell was recorded. The RNA were extracted and the cDNA libraries were built according to the SmartSeq3xpress protocol.
2023-10-10 | E-MTAB-12325 | biostudies-arrayexpress
Project description:We want to investigate how cells in the specific zones in murine liver are affected by age-related changes of the microenvironment. To this end, we generated high-quality scRNA-seq dataset of hepatocytes using Smart-seq3express from 2 young (3-5 months) and 2 old (18-20 months) male mice. Livers were perfused and viable hepatocytes were FACS-sorted based on size. In addition, we recorded ploidy levels of hepatocytes. We retained 545 hepatocytes in total after initial filtering.
2023-11-01 | E-MTAB-12579 | biostudies-arrayexpress
Project description:A highly complex set of 264 molecular spikes, based on 11 unique spike sequences spanning different lengths (570 to 3070 nts) and GC contents (40-60%) was designed. In order to be able to precisely evaluate quantification over different expression levels, transcript lengths and GC contents, barcodes of 7 nucleotides in 2-fold abundance steps were cloned into each spike sequence (12 steps in duplicates; 24 barcodes per sequence) creating a standard curve for each spike sequence. To determine the molecular abundance of each of the 264 molecular spike-ins (i.e., the ‘ground truth’), we performed an exhaustive sequencing across the spike barcodes and spUMIs and determined the total complexity in the pool to be 76 million unique molecules
2022-02-16 | E-MTAB-11448 | biostudies-arrayexpress
Project description:Allele-sensitive RNA sequencing of single-cells can be used to infer the kinetics of transcriptional bursts in eukaryotic cells. Here, we used the Smart-seq3 protocol to prepare libraries from two 384-well plates of primary mouse fibroblasts. The fibroblasts were derived from tail explants of a male adult mouse (F1 offspring of C57 x CAST cross). The samples were sequenced to high depth using MGI's DNBSEQ G400RS platform using paired-end 100 bp reads.
2021-02-25 | E-MTAB-10148 | biostudies-arrayexpress
Project description:An increasing number of long non-coding RNAs (lncRNAs) have confirmed important functions, yet little is known about their transcriptional dynamics and it remains challenging to determine their regulatory functions. Here, allele-sensitive single-cell RNA-seq was used to demonstrate that lncRNAs have lower burst frequencies compared to mRNAs. We observed an increased cell-to-cell variability in lncRNA expression that was due to more sporadic bursting (lower frequency) with larger numbers of RNA molecules being produced. Exploiting heterogeneity in asynchronously growing cells, we identified and experimentally validated lncRNAs with cell-state specific functions involved in cell cycle progression and apoptosis. Finally, we identified cis-functioning lncRNAs and knockdown of these lncRNAs modulated either transcriptional burst frequency or size of the nearby protein-coding gene. Collectively, we identify distinct transcriptional regulation of lncRNAs and we demonstrate a role for lncRNAs in the regulation of transcriptional bursting of mRNAs.
2021-11-19 | E-MTAB-11054 | biostudies-arrayexpress
Project description:The impact of gDNA contamination on RNA-seq experiments performed with the prime-seq method was assessed by introducting artifical contamination using mouse gDNA this enables us to identify contamination by species specific assignment of mapped reads.
2021-08-01 | E-MTAB-10141 | biostudies-arrayexpress
Project description:Systemic lupus erythematosus (SLE) is an autoimmune disorder with systemic inflammation, autoantibody accumulation and organ damage. The abnormalities of double-negative (DN) T cells are considered as an important commander of SLE. Neddylation, an important type of protein post-translational modification (PTM), has been well-proved to regulate T cell-mediated immune response. However, the function of neddylation in SLE remains largely unexplored. Here, we reported that neddylation inactivation with MLN4924 or genetic abrogation of Ube2m in T cells prevented SLE development for decreased DN T cell accumulation. Further investigations revealed that inactivation of neddylation blocked Bim ubiquitination degradation and maintained Bim level in DN T cells, contributing to the apoptosis of the accumulated DN T cells for Fas mutation. Then double knockout (KO) lupus-prone mice (Ube2m-/-Bim-/-lpr) were generated and results showed that loss of Bim interrupted the improvement of DN T cell apoptosis and the consequential relieved lupus symptoms for Ube2m KO. Our findings identified that neddylation inactivation promoted Bim-mediated apoptosis of DN T cells and prevented lupus progress. Clinically, we also found the percentages of DN T cells were improved accompanied with reduced apoptosis of DN T cells in SLE patients. Moreover, the neddylation of Cullin1 was higher while Bim level was decreased in SLE patients compared with healthy control. Meantime, the inhibition of neddylation induced Bim-dependent apoptosis of DN T cells isolated from SLE patients. Together, these findings provide the first evidence of the neddylation role in lupus development, suggesting a novel therapeutic strategy for lupus.
2024-01-26 | PXD045686 | Pride