Single-cell sequencing of HEK293FT cells with molecular spikes and recorded cell size
Ontology highlight
ABSTRACT:
INSTRUMENT(S): DNBSEQ-G400
ORGANISM(S): Homo sapiens
SUBMITTER: Christoph Ziegenhain
PROVIDER: E-MTAB-11433 | 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: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: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:Plate-based single-cell RNA-sequencing methods with full-transcript coverage typically excel at sensitivity but are more resource and time-consuming. Using miniaturized and streamlined Smart-seq3xpress protocol, we sequence >26,000 human peripheral blood mononuclear cells to generate a highly granular gene- and isoform-level atlas.
2022-05-16 | E-MTAB-11452 | biostudies-arrayexpress
Project description:Molecular characterization of perinatal liver HSCs.
2023-09-30 | E-MTAB-13293 | biostudies-arrayexpress
Project description:Molecule counting is central to single-cell sequencing, yet no experimental strategy to evaluate counting performance exist. Here, we introduce RNA spike-ins containing inbuilt unique molecular identifiers (molecular spikes) that we use to monitor single-cell RNA counting performance across methods and to identify experimental steps essential for accurate counting. In this dataset, we add molecular spikes to popular single-cell RNA-seq protocols: SCRB-seq, Smart-seq3 and 10x Genomics (v2). For SCRB-seq and Smart-seq3, we also include variations of the library preparation procedure that are suspected to lead to changes in the UMI counting accuracy.
2022-02-16 | E-MTAB-10372 | biostudies-arrayexpress
Project description:Microarray measurements are susceptible to error caused by cross-hybridization and bulk hybridization. To characterize the effect of these off-target interactions, we analyzed samples made from baseline (S. cerevisiae) alone or mixed with a high-concentration 1kb spike. The spike is a 1 kb sequence amplified from the Schizosaccharomyces pombe gene SPAC27D7.10c, with an added oligo(dT)-T7 promoter. The baseline and spike samples were mixed at the double-stranded cDNA stage.
2010-03-21 | GSE16980 | GEO
Project description:Microarray measurements are susceptible to error caused by cross-hybridization and bulk hybridization. To characterize the effect of these off-target interactions, we analyzed samples made from baseline (S. cerevisiae) alone or mixed with a high-concentration 1kb spike. The spike is a 1 kb sequence amplified from the Schizosaccharomyces pombe gene SPAC27D7.10c, with an added oligo(dT)-T7 promoter. The baseline and spike samples were mixed at the double-stranded cDNA stage. Three arrays: 100% baseline, 99% baseline + 1% spike, 90% baseline + 10% spike. There are no replicates.
2010-03-21 | E-GEOD-16980 | biostudies-arrayexpress
Project description:The identification of the genetic risk factors in patients with isolated cleft palate by whole genome sequencing analysis. Pathogenic or likely pathogenic variants were discovered in genes associated with CP (TBX22, COL2A1, FBN1, PCGF2, and KMT2D) in five patients; hence, rare disease variants were identified in 17% of patients with non-syndromic isolated CP. Our results are relevant to routine genetic counselling practice and genetic testing recommendations.
2022-03-30 | E-MTAB-11360 | biostudies-arrayexpress
Project description:We investigated a novel, simple approach to induce the production of cryptic secondary metabolites in actinomycetes by stimulating the organism with high-intensity monochromatic green light (180 radiation unit). Streptomyces coelicolor A3(2) produces blue antibiotic actinorhodin (ACT) and red antibiotic undecylprodigiosin (RED). Using these two pigment antibiotics as indicators, we found that sporulation acceleration and regulation of the antibiotic production pathways can be induced by using high-intensity monochromatic green LEDs. Therefore, we investigated the immediate response of S. coelicolor A3(2) gene expression to the strong green LED stimulation.
2023-12-10 | E-MTAB-12876 | biostudies-arrayexpress