Dataset Information

A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells.

ABSTRACT: RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3' or 5' termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

SUBMITTER: Haile S

PROVIDER: S-EPMC8209500 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

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).

Dataset Information

A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets