技術概述
Illumina single cell technology uses particle-templated instant partitions (PIPs) to barcode and segregate single cells for RNA sequencing.
Without the need for microfluidics device, individual cells are captured gently in an emulsion by simple vortexing, followed by cDNA synthesis and library preparation. The library will undergo next generation sequencing and data analysis to reveal gene expression profile at single-cell resolution.
With simple workflow and minimal instrumentation, Illumina single cell technology enables scalable single cell capture and increases cost efficiency. Our Core currently provides Illumina Single Cell 3’ RNA Prep service. Please feel free to contact us for service details.
服務費和訂購
服務價格
The Core offers 3’-end gene expression profiling of up to 10,000 cells per sample. Full service from cell capture, cDNA synthesis, subsequent library preparation, Illumina sequencing to data analysis by DRAGEN are available. Please contact Dr. KWOK, Hin (pipsinglecell.cpos@hku.hk / 2831-5483) 用於專案討論和服務報價。.
價格受以下因素影響：
- 標靶細胞數量（影響定序成本）
- 生物資訊分析需求
- Affiliated institute
服務訂購
請參考 樣品要求及提交 樣品要求部分。.
請透過以下方式提交服務請求 iLab.
技術細節
Cell Capture and Library Preparation
Cells in suspension are first mixed with barcoded hydrogel template particles and oil. By vortexing, cells are instantaneously captured in particle-templated instant partitions (PIPs). They are then lysed to release their mRNA, which are in turn captured by poly(dT) primers on the barcoded beads. Subsequently, the emulsion is broken and washed to recover the beads for reverse transcription.
Next, full length cDNA is fragmented, ligated with adapters and amplified into libraries in bulk. Final libraries will contain unique cell barcodes and intrinsic molecular identifiers (IMIs) for more accurate cell and molecular counting.
Source: Illumina
定序運行
Libraries generated are compatible with Illumina sequencers. In order to read the transcript sequences on one end, and the barcode on the other end, paired-end sequencing reads are required. Sequencing depth varies based on user application needs but vendor recommends to start with a depth of 20,000 read pairs per input cell.
工作流程
樣品要求及提交
樣品要求
Sample Type:
Single cell/nuclei suspension (Fresh / Fixed*)
Cell Size:
60 μm or smaller, well dissociated without clumps or doublets
Concentration:
3,400 cells/μl, minimum 50 μl
Viability:
>90%
Sample Buffer:
Cell Suspension Buffer (Illumina)**
* Fixed cells / nuclei need to be generated by DSP-Methanol Fixation. Alternative fixation approaches (e.g. formaldehyde) have not been validated and are not recommended.
**It is critical to complete the Cell Suspension Buffer wash of cell suspension prior to input into the PIPs to ensure any inhibitory reagents (e.g. FBS, BSA > 1%, high salt or calcium, carryover organic solvents) are sufficiently washed out of the cell suspension. The core will perform one wash step to change the buffer to Cell Suspension Buffer prior to mixing with PIPs.
樣品提交
由於工程性質特殊，樣品提交後需立即處理。 請提前聯繫我們的同事。 （透過電話/電子郵件顯示） 在此用於時段預約。.
After sample submission date and details are confirmed, please submit service request through iLab. Please submit Single Cell (Illumina) sample submission form 作為附件 iLab.
服務條款
從提交樣本到交付定序數據，全套服務的預期週轉時間通常為 2-4 週。週轉時間可能因訂單需求而有所不同。.
聯絡我們
郭欣醫生
李女士，瑞秋
