GSM4629065: Single-cell RNA-Seq of Tumor-infiltrating T cells; Mus musculus; RNA-Seq
The single-cell libraries were prepared using the Flow-cell devices composed of multiple vertical flow array chips) (VFACs, Shirai et al, Scientific Reports, 6, srep36014, 2016). VFAC (1 mm square) contains 100 microchambers packed with 1~2 x105 magnetic beads (1 µm in diameter) immobilized 7.5~15 x109 RT probes. RT probes consisted of 18nt of poly-T sequence, 6 nt of cell identifier (Cell-ID), 7nt of random sequence as UMIs(Unique Molecular Identifiers), and 30nt of common sequence (CS) for amplification (Supplementary Table 2). The procedures from cell captureing to cDNA synthesis were performed in the flow-cell device. In brief summary, FACS-sorted tumor-infiltrating lymphocytes isolated from YTN2 and YTN16 tumor tissues were washed once with PBS, and resuspended in PBS containing 90 copies/μL of spiked cRNA to a final concentration of 80 cells/μL. After adding 80 cells per a VFAC, the flow-cell device was connected to a vacuum pump (Ulback KOKI, Inc.) for appling negative pressure to the rear side of the VFACs, and single-cells were captured onto laser 4-micro-meter holes in diameter fabricataed at top of the microchambers respectively. Following cell lysis reagent, 4.5 μL of RT reagent (a mixture of 1.0 μL of 5 x FS buffer, 1.0 μL of 100 mM DTT, 1 μL of 10 mM dNTPs, 0.3 μL of 10 % Tween20, 0.4 μL of RNase OUT, and 0.4 μL of Super Script III (Thermo Fisher Scientific)) was added per a VFAC in the flow-cell device, and reverse transcription was performed for 50 minutes at 50 ℃ in a thermostatic incubator. Each VFAC was took out from the flow-cell device into a 0.2 mL tube containing 100 μL of resuspension buffer (50 mM Tris (pH 8.0) containing 0.1% Tween 20). Magnetic beads on which single-cell cDNA libraries were constructed were collected from microchambers of VFAC using a neodymium magnet, and washed twice with 50 μL of resuspend buffer. After exonuclease I treatment, 1st PCR was performed with primers listed in Supplementary Table 3, and the product was purified with Ampure XP (Beckman Coulter). Following 2nd PCR was performed with primers listed in Supplementary Table 4, the product was purified with Ampure XP. 3rd PCR was performed with primers containing illumina-tag sequences and index (chip-ID) listed in Supplementary Table 5, and the product was purified with Ampure XP. The single-cell libraries were analyzed with 2100 Bioanalyzer high sensitivity DNA kit (Agilent), to confirm a fragment distribution within a size range of 461–667 bp.  More than 3M reads for each chip was assigned to get sufficient deep sequencing data. Libraries of 3 or 4 chips were pooled before sequencing. Infomations of cells on each chip were shown in Supplementary Table 6. The final concentration of each 3rd PCR product should be diluted with 10 mM Tris:HCl (pH7.5) with 0.1 % tween20 to be 3.2 nM. According to the user manual of Miseq, denature is carried out. Phi X control is mixed at 10%. Paired-end sequencing (read 1 = 60bp, read 2 = 90bp) was carried out using Miseq (150 cycle (v3) reagents).
GEO Accession
GSM4629065