GSM6543627: Liver, DDC diet, 0 days, n=1  for all normal diet in Smart_seq2 method [Smart_seq2_Hepatocytes_Z101]; Mus musculus; RNA-Seq
We collected primary hepatocytes via perfusion as reported from normal (Rosa26-mTmG mice for denoting normal liver-derived cells based on mTomato expression, n=2) and DDC-injured (WT mice, n=2) liver, and performed pooled 10X Chromium scRNA-seq (10X genomics Methods). In Smart-seq2 protocols, we used Sox9-EGFP mice to trace hepatocyte-derived Sox9-positive LPLC in injured liver (n=2 for each group). Injured hepatocytes and LPLC from DDC-injured livers were isolated by sorting GFP- or GFP+ hepatocytes by the BD influx cell sorter based on size/granularity and GFP expression. We also isolated and enriced macrophages via  anti-F4/80 (Ebioscience, 13-4801-82, 1:200) staining. After MACS based on anti-Biotin Microbeads (Miltenyi, 130-105-637) using AutoMACS Pro for enriched macrophages, 10,210 non-parenchymal cells from normal livers (Rosa26-mTmG mice, n=2) and DDC-injured (WT mice, n=2) livers were pooled and sequenced on Novaseq lanes by Novagene AIT. For 10X pipeline, isolated hepatocytes or enriched macrophages were processed using the Chromium Single Cell 30 (v3 Chemistry) platform (10X Genomics, Pleasanton, CA). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. For Smart-seq2, single hepatocyte was placed into the lysis buffer by the mouth pipette. The reverse transcription reaction was performed with 25 nt oligo (dT) primer anchored with an 8 nt cell-specific barcode and 8 nt unique molecular identifiers (UMI). After the first-strand synthesis, the second-strand cDNA was synthesized, and the cDNA was amplified by 14 cycles of PCR. The amplified cDNA of single cells was then pooled together for the following steps. Biotinylated pre-indexed primers were used to further amplify the PCR product by an additional 4 cycles of PCR to introduce biotin tags to the 3' ends of the amplified cDNAs. Approximately 300 ng cDNA was sheared to approximately 300 bp by Covaris S2, and the 3' terminal of the cDNA was captured by Dynabeads® MyOne Streptavidin C1 beads (Thermo Fisher). The RNA-seq library was constructed using a Kapa Hyper Prep Kit (Kapa Biosystems) and subjected to 150 bp paired-end sequencing on an Illumina HiSeq 4000 platform (sequenced by Annoroad).