S1000 touch thermal cycler

Single cell suspensions (300–500 living cells per micro liter) were loaded on a Chromium Single Cell Controller (10x Genomics) to generate single-cell Gel Bead-In-Emulsions (GEM) by using single cell 3 ’Library and Gel Bead Kit V3 (10x Genomics, 1000075) and Chromium Single Cell B Chip Kit (10x Genomics, 1000074). In short, single cells were suspended in PBS containing 0.04% BSA. About 6,000 cells were added to each channel, and the target cell will be recovered was estimated to be about 3,000 cells. Captured cells were lysed and the released RNA was bar-coded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 min, followed by 85°C for 5 min, and hold at 4°C. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200 (performed by CapitalBio Technology, Beijing). Single-cell RNA sequencing libraries were constructed using Single Cell 3’Library and Gel Bead Kit V3. The libraries were finally sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing).

After first-strand synthesis, the RT master mix in each well was replaced with KOH (0.08 M, 75 μL). After incubation at room temperature for 5 min, the slices were washed with EB buffer (100 µL), and then Second Strand Mix (75 μL) was added for second-strand synthesis. cDNA amplification was performed on a S1000TM Touch Thermal Cycler (Bio-Rad). Visium spatial libraries were constructed using the Visium spatial library construction kit (10x Genomics, PN-1000184) according to the manufacturer’s instructions. The final libraries were sequenced using an Illumina NovaSeq6000 sequencer with a sequencing depth of at least 100,000 reads per spot using 150 bp (PE150) read strategy (performed by CapitalBio Technology, Beijing).

Using single-cell 5′ Library and Gel Bead Kit (10x Genomics, 1000006) and Chromium Single Cell A Chip Kit (10x Genomics, 120236), the cell suspension (300–600 living cells per microliter determined by Count Star) was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. In short, single cells were suspended in PBS containing 0.04% BSA. Then the cells were added to each channel, and the target cell will be recovered. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs [23 (link)]. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53 °C for 45 min, followed by 85 °C for 5 min, and hold at 4 °C. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200 (performed by CapitalBio, Beijing).

Each 10,000 nuclei were used for the snRNA or snATAC library construction.
For snRNA-seq, the single cell 3′ GEM, Library & Gel Bead Kit V3.1 (10× Genomics, 1000075) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074) were used. To generate single-nuclei gel beads in emulsion, the nuclei suspension was loaded onto the Chromium single cell controller (10× Genomics). Then suspended the single nuclei in PBS (containing 0.04% BSA). Captured cells were lysed to release their RNA and barcoded through reverse transcription in individual GEMs. The reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio-Rad) at 53 °C for 45 min, followed by 85 °C for 5 min. The cDNA was kept at 4 °C and then amplified for sequencing.
For snATAC-seq, incubating the nuclei with Tn5 transposase. Then the nuclei suspension was loaded into the Chromium microfluidic chip E with 10x Genomics reagents and barcoded with a 10x Genomics Chromium Controller (10x Genomics, Pleasanton, CA). DNA fragments were subsequently amplified, and the sequencing libraries were constructed with reagents from a Chromium Single Cell ATAC reagent kit (10x Genomics; PN-1000110, PN-1000156, PN-1000084) according to the manufacturer’s instructions. After preliminary quantification and quality inspection, libraries were then pooled and loaded on an Illumina NovaSeq with 2 × 50 paired-end kits.

Single Cell G Chip Kit (10x Genomics, 1000120), the cell suspension (300-600 living cells per microliter determined by Count Star) was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 min, followed by 85°C for 5 min, and hold at 4°C. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200 (performed by CapitalBio Technology, Beijing). Single cell RNA-Seq library preparation According to the manufacture's introduction, Single-cell RNA-seq libraries were constructed using Single Cell 3' Library and Gel Bead Kit V3.1. The libraries were finally sequenced using an Illumina Novaseq 6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (sPtrEa1t5e0gy) (performed by CapitalBio Technology, Beijing).

Nuclei were loaded onto a Chromium single-cell controller (10x Genomics) to generate single-nucleus gel beads in the emulsion (GEM) using a single-cell 3' Library and Gel Bead Kit V3.1 (10x Genomics, 1000075) and Chromium Single Cell B Chip Kit (10x Genomics, 1000074) according to the manufacturer’s instructions. Approximately 8040 nuclei were captured from each sample. The captured nucleus was lysed, and the released mRNA was barcoded through reverse transcription in individual GEMs. Reverse transcription was performed to generate cDNA using an S1000TM Touch Thermal Cycler (Bio-Rad) with the following parameters: 53°C for 45 min, 85°C for 5 min, and 4°C until further use. The cDNA was then amplified, and the quality was assessed using an Agilent 4200 (CapitalBio Technology).