GSM4971880: DS_MM_D0_ctrl_4; Mus musculus; RNA-Seq
the single-cell transcriptomes are generated with a modified version of the Drop-seq protocol using Igepal CA-630 for cell lysis [Purification of mouse and human PBMCs]  Mouse and human lymphocytes were isolated from a mouse spleen and human blood with the use of Lympholyte M (Cedarlane) and Ficoll-Paque PLUS (GE Healthcare) density gradient media respectively. Cells were washed with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity)  where individual cells meeting the gating strategy were sorted in a tube containing media and used immediately for generating Drop-seq single-cell transcriptomes. [Purification of naive P14 T cells]  Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 Î¼m nylon cell strainer (BD Falcon) and lysing red blood cells with a hypotonic ACK buffer. NaÃ¯ve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech  Bergisch-Gladbach  Germany). Surface staining was performed for 40 min at 4Â°C in supplemented DMEM media (Gibco  ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7  eBioscience)  CD4 (RM4-4  Biolegend)  and VÎ±2 (V20.1  eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq)  which was subsequently spun down  snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar). [Purification of activated P14 T cells]  Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 Î¼m nylon cell strainer (BD Falcon) and red blood cells were lysed with a hypotonic ACK buffer. NaÃ¯ve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech  Bergisch-Gladbach  Germany). 2x103 CD45.1+ P14 TCRÎ±Î² were transferred into naÃ¯ve CD45.2+ C57BL/6 mice  followed by their infection. On the day of the sort  single cell splenocyte suspensions were obtained as described above. Activated transgenic P14 CD8 T-cells were isolated using anti-CD45.1 biotin/anti-biotin conjugated microbeads and magnetic MACS cell separation (Miltenyi Biotech  Bergisch-Gladbach  Germany). Surface staining was performed for 40 min at 4Â°C in supplemented DMEM media (Gibco  ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7  eBbioscience)  CD4 (RM4-4  Biolegend)  CD45.1 (A20  eBioscience) and CD45.2 (104  eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq)  which was subsequently spun down  snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar). [Drop-seq library construction protocol]  The original Drop-seq protocol was performed as described by Macosko and colleagues16. All runs were performed at 3000 Âµl/hour cell and bead rates with the use of Drop-seq PDMS device (Nanoshift). The following reagents were used for the Drop-seq modification described in this article: Lithium dodecyl sulfate (L9781  Sigma-Aldrich)  Lithium chloride (62476  SigmalAldrich)  Igepal (I8896-50ML  Sigma-Aldrich)  NaCl (S3014  Sigma-Aldrich)  3âLNA(G)TSO (IDT)  Maxima H Minus RT (EP0753  ThermoFisher)  SuperScript IV RT (18090010  ThermoFisher)  SMARTScribe RT (639536  Takara Bio) and Ficoll PM 400 (GE17-0300-10  GE Healthcare). After RNA purification and reverse transcription  the generated single-cell cDNA was amplified for 18 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads. 1 ng of the resulting amplified cDNA were used for library preparation with the Illumina Nextera XT DNA Library reagents (FC-131-1024  Illumina). After PCR amplification of the fragmented libraries  the samples were purified with (0.6x) Agencourt AMPure XP beads and eluted in 10 Âµl of molecular grade water. The quality of the resulting library was assessed with the use of Agilent High Sensitivity DNA Kit (5067-4626  Agilent). The library quantification was performed based on the Illumina recommendations (SY-930-1010  Illumina) with the use of KAPA SYBR FAST qPCR Master Mix (KK4600  Kapa Biosystems). The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run  paired-end  20 bp read 1  45 bp read 2  single-indexed sequencing resulting in 0.5 million reads per single-cell. [SCRB-seq library construction protocol]  The original SCRB-seq protocol was performed as described by Soumillon and colleagues17. To allow profiling of T cells  we introduced a step of RNA purification before reverse transcription with the use of RNAClean XP (A63987  Beckman Coulter). This was used for all modifications assessed in this paper. The following reagents were used for the SCRB-seq modification described in this article: Triton X-100 (T9284-100ML  Sigma-Aldrich)  Buffer TCL (1031576  Qiagen)  2-Mercaptoethanol (M3148-25ML  Sigma-Aldrich)  3âLNA(G)TSO (IDT) and Betaine solution (B0300-1VL  Sigma-Aldrich). After reverse transcription  the single-cell transcriptomes were amplified without pooling for 20 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads and libraries were generated as for Drop-seq. The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run  paired-end  16 bp read 1  49 bp read 2  single-indexed sequencing resulting in 0.5 million reads per single-cell.