title: NextSeq 550 paired end sequencing; Single-cell RNAseq of Human T lymphocyte derived from H7N9 infected patients
design: Single-cell RNAseq of Human T lymphocyte derived from H7N9 infected patients
library_construction_protocol: Patients were selected based on: 1) comparable time-points; but differential disease outcomes: one patients was from the fatal outcome group, one patient from survival group. Peripheral Blood Mononuclear Cells were separated from Whole blood using density gradient centrifugation and used directly for staining and sorting. Cells were stained with antibodies: anti-CD3-PB (BD Cat #558117, UCTH1, 1:200), anti-CD8-FITC (BD Cat #347313, SK1, 1:50), anti-CD27-PE-Cy7 (eBioscience Cat #25-0279-42, O323, 1:25), anti-CD45RA-APC-Cy7 (BD Cat #560674, HI100, 1:50), anti-HLA-DR-ECD (Beckman Coulter Cat#IM3636, Immu-357, 1:25), anti-CD38-PerCPCy5.5 (Biolegend, Cat#303522, 1:50), anti-PD-1-APC (Biolegend #329908, EH12.2H7, 1:25) and Live/Dead-aqua 525 (Invitrogen, 1:800). Lymphocytes were washed, acquired on an Aria II  sorter and single cell sorted into 96 well plates which already contains 2ul of lysis buffer. a33 has two time-points after disease onset:  d12 and d31;   a11 has two time-points after disease onset:  d15  and d29.    PBMC samples were taken out from liquid nitrogen and placed on dry ice, thawed in a 37 degree waterbath and transferred into 9ml cRPMI, and then washed at 500g, 7min, then resuspended in 50ul of Live/Dead-aqua 525 (Invitrogen, 1:800) in PBS, and then incubated for 15min at dark, washed and resuspended with 50ul of antibodies mixture : anti-CD3-PB (BD Cat #558117, UCTH1, 1:200), anti-CD8-FITC (BD Cat #347313, SK1, 1:50), anti-CD27-PE-Cy7 (eBioscience Cat #25-0279-42, O323, 1:25), anti-CD45RA-APC-Cy7 (BD Cat #560674, HI100, 1:50), anti-HLA-DR-ECD (Beckman Coulter Cat#IM3636, Immu-357, 1:25), anti-CD38-PerCPCy5.5 (Biolegend, Cat#303522, 1:50), anti-PD-1-APC (Biolegend #329908, EH12.2H7, 1:25) for 30min, wash with MACS buffer for twice and then single sorted on Aria II . Single cell RNA sequencing was performed using Smart-seq2 as described by Picelli et al. (Picelli et al. Nature methods 2013) with the following modifications. Cell lysis buffer was prepared by adding 1 μlRNase inhibitor (Clontech, Mountain View, CA) to 19 μlTriton X-100 solution (0.2% v/v). Cells were sorted directly into tube containing 2 μl lysis buffer, 1 μl dNTP mix (10mM) and 1 μl oligo-dT primer at 5 μM. Tubes were stored at -80 °C until needed. Reverse-transcription and PCR amplification were performed as described with the exception of reducing the IS PCR primer to a 50 nM final concentration and increasing the number of cycles to 28. Sequencing libraries were prepared using the Nextera XT Library Preparation Kit (Illumina; San Diego, CA, USA) and sequencing was performed on Illumina NextSeq sequencing platform. ERCC RNA Spike-In Mix 1 was utilised, from ThermoFisher, cat 4456740 (1:400,000,000 dilution)
attributes.tag: Experimental Factor: clinical history
attributes.value: fatal group
attributes.tag: Experimental Factor: sampling time point
attributes.value: 12