Single-cell barcoding and sequencing using droplet microfluidics

Abstract

Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile >75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.

Clonal analysis of lineage fate in native haematopoiesis

“…Transcriptome barcoding and preparation of libraries for single-cell mRNA-sequencing was performed using the most up-to-date inDrops protocol 34 . For our experiment, the Lin-Sca1+cKit+ BM fraction from a single BL6 mouse was labelled and FACS sorted to purify the entire LT-HSC, MPP1, MPP2, MPP3 and MPP4 fractions.…”

Comparative analysis of droplet-based ultra-high-throughput single-cell RNA-seq systems

“…This droplet strategy greatly elevates the reaction throughput and dramatically reduces cost. Currently, there are three prevalent droplet-base systems for high-throughput scRNA-seq, namely inDrop Klein et al, 2015;Wagner et al, 2018;Zilionis et al, 2017), Drop-seq 4 (Farrell et al, 2018;Macosko et al, 2015) and 10X Genomics Chromium (10X) . All of them have been demonstrated to be robust and practical in generating cDNA libraries for thousands of cells in a single run with acceptable cost.…”

Формирование Гидрогелевых Частиц Из Полиакриламида И PEGDA В Микрофлюидном Генераторе Эмульсий С Фокусировкой Потока

“…Они рассматривают-ся в качестве перспективных кандидатов для создания таргетных средств доставки лекарственных препара-тов [1,2], кодирования фрагментов ДНК при секвениро-вании генома отдельных клеток [3], специфических мар-керов для обнаружения и выделения онкомаркеров [4,5], микрореакторов для проведения генетических иссле-дований методом цифровой капельной полимеразной цепной реакции (ПЦР) [6], упаковки отдельных клеток для регенерации поврежденных тканей [7].…”