Chromium single cell controller instrument

scRNA-seq libraries were prepared with Chromium Single cell 3’ Reagent v3 Kits according to the manufacturer’s protocol. Single-cell suspensions were loaded on the Chromium Single Cell Controller Instrument (10× Genomics) to generate single-cell gel beads in emulsions (GEMs). Briefly, about 2×10⁵ PBMC single cells were suspended in calcium- and magnesium-free PBS containing 0.04% weight/volume BSA. About 22,000 cells were added to each channel with a targeted cell recovery estimate of 10,000 cells. After generation of GEMs, reverse transcription reactions were engaged barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, New York, NY, USA). cDNA was then amplified by PCR with appropriate cycles which depend on the recovery cells. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed, index adaptor ligated, and library amplification. Then these libraries were sequenced on the Illumina sequencing platform (NovaSeq6000) and 150 bp paired-end reads were generated. The GEM generation, library construction, and sequencing were performed by OE Biotech Co., Ltd (Shanghai, China).

The scRNA-seq libraries were prepared using Chromium Single Cell 3 Reagent v3 Kits. Single-cell suspensions were loaded on a Chromium Single Cell Controller Instrument (10X Genomics) to generate single-cell gel beads in emulsions (GEMs). Briefly, about 16,00020.000 cells were added to each channel, with a targeted cell recovery estimate of 5,0008,000 cells. After generation of GEMs, single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3Library& Cell Bead Kit (10X Genomics) according to the manufacturers protocol. Libraries were sequenced with an IlluminaNovaseq6000 using high-output 75-cycle kits with apreviously reported read length configuration (21 (link)).

Single-cell barcoding and scRNA-seq library preparation were performed based on 10X Genomics single-cell RNA sequencing platform (10×Genomics, Pleasanton, CA, USA). Before barcoding, the cell concentrations of single-cell suspensions were counted using a hemocytometer (TC20, Bio-Rad, Hercules, CA, USA) and adjusted to 1,000 cells/μl. Single-cell suspensions were loaded on the Chromium Single Cell Controller Instrument (10× Genomics, Pleasanton, CA, USA) to generate single cell gel beads in emulsions (GEMs), and 10×Genomics Chromium barcoding system was adopted to construct 10×barcoded cDNA library according to the manufacturer’s instructions. All libraries were sequenced on the Illumina HiSeq X Ten sequencing platform (Illumina, San Diego, CA, USA) with 150 bp pair-end module.

Brains from healthy mice (n = 8), ischemic stroke mice (n = 8), or hemorrhagic stroke mice (n = 8) were rapidly removed, and target tissues were carefully collected and dissociated using an adult brain dissociation kit from Miltenyi Biotec (Bergisch Gladbach, Germany). Subsequent tissue processing and data acquisition were performed by Oebiotech (Shanghai, China). Briefly, single-cell gel beads in emulsions (GEMs) were generated by loading single-cell suspensions onto a Chromium Single-Cell Controller Instrument (10X Genomics). Approximately 12,000 cells were added to each channel. After that, reverse transcription reactions were engaged to generate barcoded full-length cDNA, and cDNA clean-up was performed with DynaBeads Myone Silane Beads (Thermo Fisher Scientific). Next, cDNA was amplified by PCR and the amplified cDNA was fragmented, end-repaired, A-tailed, and ligated to an index adaptor, and then the library was amplified. Every library was sequenced on a HiSeq X Ten platform (Illumina), and 150 bp paired-end reads were generated. the scRNA-seq analysis was performed in one batch or in several batches.

The Chromium Single-Cell 3ʹ Library and Gel Bead KIT V3 (10× Genomics, 1000075) were used to prepare barcoded scRNA-seq libraries according to the manufacturer’s protocol. Single-cell suspensions were loaded onto a Chromium Single-Cell Controller Instrument (10× Genomics) to generate single-cell gel beads in emulsions (GEMs), according to the manufacturer’s protocol. Approximately 8000 cells were added to each channel to capture 5000 cells per library. The captured cells were lysed, and the released RNA was barcoded through reverse transcription in individual GEMs. Using an S1000TM Touch Thermal Cycler (BioRad) to reverse transcribe, the GEMs were programmed at 53°C for 45 min and 85°C for 5 min and held at 4°C. cDNA was generated and then amplified, and the quality was evaluated using Agilent 4200. Each library was sequenced on an Illumina NovaSeq 6000 sequencer with a sequencing depth of at least 100,000 reads per cell, and 150 bp (PE150) paired-end reads were generated (performed by CapitalBio, Beijing).