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SRX892948: GSM1620350: PS034_R2_0_155; Homo sapiens; RNA-Seq
1 ILLUMINA (NextSeq 500) run: 262,834 spots, 13.7M bases, 7.7Mb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: Scalable Microfluidics for Single Cell RNA Printing and Sequencing
show Abstracthide Abstract
Single cell transcriptomics has emerged as a powerful approach to dissecting phenotypic heterogeneity in complex, unsynchronized cellular populations. However, many important biological questions demand quantitative analysis of large numbers of individual cells. Hence, new tools are urgently needed for efficient, inexpensive, and parallel manipulation of RNA from individual cells. We report a simple microfluidic platform for trapping single cell lysates in sealed, picoliter microwells capable of “printing” RNA on glass or capturing RNA on polymer beads. To demonstrate the utility of our system for single cell transcriptomics, we developed a highly scalable technology for genome-wide, single cell RNA-Seq. The current implementation of our device is pipette-operated, profiles hundreds of individual cells in parallel with library preparation costs of ~$0.10-$0.20/cell, and includes five lanes for simultaneous experiments. We anticipate that this system will ultimately serve as a general platform for large-scale single cell transcriptomics, compatible with both imaging and sequencing readouts.!Series_type = Expression profiling by high throughput sequencing Overall design: A microfluidic device that pairs sequence-barcoded mRNA capture beads with individual cells was used to barcode cDNA from individual cells which was then pre-amplified by in vitro transcription in a pool and converted into an Illumina RNA-Seq library. Libraries were generated from ~600 individual cells in parallel and extensive analysis was done on 396 cells from the U87 and MCF10a cell lines and from ~500 individual cells with extensive analysis on 247 cells from the U87 and WI-38 cell lines. Sequencing was done on the 3''-end of the transcript molecules. The first read contains cell-identifying barcodes that were present on the capture bead and the second read contains a unique molecular identifier (UMI) barcode, a lane-identifying barcode, and then the sequence of the transcript.
Sample: PS034_R2_0_155
SAMN03379472 • SRS859446 • All experiments • All runs
Organism: Homo sapiens
Library:
Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: RNA was extracted from individual cells in individual microfluidic chambers following cell lysis by Triton X-100 and freeze-thaw. mRNA from individual cells was reverse transcribed with a primer containing a cell-identifying barcode followed by oligo(dT). Following second strand synthesis using DNA Polymerase I and reagents from the MessageAmp II kit (Ambion), ds-cDNA from all barcoded individual cells was pre-amplified by in vitro transcription using T7 RNA polymerase in a pool. The pools of amplified RNA from each lane of the microfluidic device were individually reverse transcribed using barcoded random hexamers containing both a unique molecular identifier (random 8-base barcode) followed by a lane-identifying barcode (6-base barcode). Illumina adapters were inserted on either end of the library during the two previous reverse transcription steps and were used to then enrich the library by PCR. The pooled library was sequenced on an Illumina NextSeq 500.
Experiment attributes:
GEO Accession: GSM1620350
Links:
External link:
Runs: 1 run, 262,834 spots, 13.7M bases, 7.7Mb
Run# of Spots# of BasesSizePublished
SRR1821351262,83413.7M7.7Mb2015-05-21

ID:
1283371

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