Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Commercially obtained fresh human hepatocytes were stained with the following primary antibodies, CD235a (349104, FITC, mouse; Biolegend), EpCAM (324208, APC, mouse; Biolegend) and ASGPR1 (563655, PE, mouse, BD Pharmingen™), all at 1:100 dilution and incubated for 30mins at 4˚C. DAPI (D1306, ThermoFisher Scientific) at 1:1000 dilution was used for live/dead staining. Briefly, cells were sorted using a BD FACS Aria II instrument and deposited as single cells into 96-well plates, pre-loaded with lysis buffer (1% Triton X-100, 1mM dNTP, 1μM oligo-dT30, 1:1.2x106 ERCC ExFold RNA spike-in, Recombinant RNase Inhibitor (2313B, Takara Clontech). Single-cell sequencing was performed using SmartSeq2. RNA was converted into cDNA using SMARTScribe Reverse Transcriptase (639538, Takara Clontech) and amplified for 21 cycles (Kapa HiFi HotStart ReadyMix 2x, KK2602, KAPA Biosystems). Successful single cell libraries were identified by capillary gel electrophoresis (DNF-474-1000, High Sensitivity NGS Fragment Analysis Kit, AATI) and converted into sequencing libraries using a Nextera XT DNA Sample Preparation Kit (FC-131-1096, Illumina). Barcoded libraries were pooled and subjected to 75 base pair paired-end sequencing on a Illumina HiSeq 2500 instrument.