Fluidigm c1 single cell auto prep system

The Fluidigm C1™ Single-Cell Auto Prep System (Fluidigm, California, USA) together with IFC plates (17 to 25 μm in size) were used to capture single cells. The Clontech® SMARTer® technology (Takara Bio, USA, Inc.) was used for the lysis, reverse transcription, and amplification of the resulting complementary DNA (cDNA), as per the manufacturer's instructions.

A sgRNA screen library was prepared and transduced into the K562-Cas9 cell. A small amount of cells was collected to demonstrate the application on the C1 platform.
The single-cell sequencing libraries were prepared using the Fluidigm C1 Single-Cell Auto Prep System, with C1 Single-Cell Auto Prep IFC for mRNA seq (Fluidigm #100–5760). The cell was prepared following the C1 protocol. The cDNA collected from the microfluidic chip was purified via QIAquick Nucleotide Removal Kit (QIAGEN# 28306). For each individual cell, 1 ng purified product was used for library preparation via the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme #TD503). The final libraries were enriched by a 0.7–1.5× double-sided selection with AMPure XP beads and eluted in 25 μL nuclease-free H₂O.

After flow sorting, single cells were captured on the Fluidigm C1 Single-Cell Auto Prep System (C1), lysed on chip, and subjected to reverse transcription and cDNA amplification using the SMARTer Ultra Low Input RNA Kit for C1 System (Clontech, Mountain View, CA). Sequencing libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) according to C1 protocols (Fluidigm). Barcoded libraries were pooled and quantified using a Qubit Fluorometer (Thermo Scientific Life Technologies, Grand Island, NY). Single-read sequencing of the pooled libraries was carried out either on a HiScanSQ or a HiSeq2500 sequencer (Illumina) with 100-base reads, using TruSeq v3 Cluster and SBS kits (Illumina) with a target depth of >2.5 M reads. Sequences were aligned to the UCSC Human Genome Assembly version 19, gene expression levels quantified using RSEM [28 (link)], and TPM values loaded into R [29 (link)] for analyses. See Additional file 1 for more details on data processing procedures.

Fluidigm C1™ Single-Cell Autoprep System (Fluidigm, South San Francisco, CA, USA) was used for single cell RNA-seq. In the initial experiment, shLuc#1 or shCTCF#1 were uploaded to a C1 integrated fluidics circuit (IFC) for cell capture, respectively. We checked the IFC to count the number of captured cells, and to distinguish between live and dead cells for later data processing. After successful completion of the second knockdown, cells from shLuc#2 and shCTCF#2 were treated similarly to the initial experiment. Single-cell RNA-seq with SMARTer protocol (Clontech, Mountain View, CA, USA) was prepared following Fluidigm manual ‘Using the C1 Single-Cell Auto Prep System to generate mRNA from Single Cells and Libraries for Sequencing’. The wells containing either zero or double cells were filtered out. We selected 24 cells with the highest quality from each IFC. The DNA materials obtained from the 96 single cells were sequenced on Illumina HiSeq 3000, as illustrated in Fig. 1c.

Single mouse lung cell capture and STA (specific target amplification) were carried out using the Fluidigm C1 Single-Cell Auto Prep System and Single-Cell Auto Prep Array integrated fluidic circuits (IFCs) (Fluidigm, South San Francisco, CA). For these experiments, medium-sized (10–17 um cell diameter) STA IFCs were used. Chip-priming, cell-loading, lysis, reverse transcription, and preamplification were performed in accordance with Fluidigm's recommended protocol using lysis and preamplification reagents from the Single Cell-to-CT Kit (Ambion/Life Technologies) and pooled preamplification primers custom designed to enrich for 96 loci of interest (20 (link)). Cells were loaded onto the chip at concentrations of 120–370 cells/μl. After capture of individual cells on the C1 chip, the isolated cells were examined by light microscopy: 261 sites were excluded because of multiple cells (258 doublets; 103 triplets) and 347 were excluded because of associated cellular debris. The remaining single cells analyzed by the C1 chip included 1107 total single cells: 207 littermate control, 169 surgical controls (including postoperative day 3 plombage and phrenic nerve controls), 265 day 1, 261 day 3, 205 day 7 cells. Cell yields varied by timepoints suggesting a changing extracellular matrix; 23 mice were studied.