title: Illumina HiSeq 2000 paired end sequencing; [Smart-seq2] scRNA-seq of mouse Plasmodium-specific CD4+ T cells
design: [Smart-seq2] scRNA-seq of mouse Plasmodium-specific CD4+ T cells
library_construction_protocol: Single PbTII cells were FACS-sorted from the spleen of one chosen donor mouse at each timepoint into wells of a 384-well plate containing cell lysis buffer. Single-cell lysates were sealed and spun at 100g for 1 min then immediately frozen on dry ice and stored at -80C. Single PbTII cells were FACS-sorted from the spleen of one chosen donor mouse at each timepoint into wells of a 384-well plate containing cell lysis buffer. The lysis buffer consisted of Triton-X, RNase inhibitor, dNTPs, dT30 primer and ERCC spike-ins (Life Technologies).  Reverse transcription and PCR were performed following the Smart-Seq2 protocol (cite Picelli paper, 2014) with the following modifications: 1µl of reverse transcription mix and 5.7µl PCR Master mix. After amplification, cDNA was subjected to quality control using 1 µl of amplified cDNA on an Agilent 2100 BioAnalyser and Agilent High Sensitiviy DNA kits (Agilent Technologies, Santa Clara, CA, ISA). 5µl of cDNA per cell was cleaned using Agencourt AMPure XP beads (Beckman Coulter UK Ltd, High Wycombe, UK) at a 1.0x ratio on a Hamilton STAR liquid handler (Hamilton Robotics, Bonaduz, Switzerland). cDNA was quantified using Biotium AccuClear High Sensitivity DNA quantification reagent and normalised to 1 ng/µl in a total volume of 500 nl before library preparation using Nextera XT DNA Sample Preparation Kit (Illumina). 125 nl of in-house index adapters (Integrated DNA Technologies, Coralville, IO, USA) similar to Illumina N7 and N5 indices were added to the tagmentation reaction before adding 1.5 µl of KAPA HiFi DNA polymerase (KAPA Biosystems, Wilmington, MA, USA) and performing 12 cycles of PCR according to the manufacturer's instructions. After PCR, all samples were pooled into 384-plex pools using a 24x16 dual indexing approach and the pool cleaned using Agencourt AMPure XP beads at a 0.6x ratio. Library pools were eluted in buffer EB and quality controlled using an Agilent 2100 BioAnalyser and Agilent High Sensitiviy DNA kits before adjusting the concentration to 4nM.