Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: 10X Genomics Chromium Controller droplet emulsion capture 10X Genomics Chromium Single-Cell 3' Gel Bead and Library V2 Kit, suspending cells in PBS with 5% FBS to a final concentration of 1 x 10^6 cells/ml (1000 cells per ul) and loaded in each channel with a target output of 3000 cells. Reactions performed with Bio-Rad C100 Touch Thermal Cycler with a 96 Deep Well Reaction Module, 12 cycles for cDNA amplificationand OCR indexing Single cells were extracted using 10X Genomics Chromium Controller droplet emulsion capture. Whole tissue RNA was purified using 1ml TRIzol reagent (4C) (Invitrogen, 15596026) added to 20mg frozen splenic tissue in a 2ml microcentrifuge tube along with a single stainless-steel bead (Qiagen, 69965) and homogenization was performed using a Tissuelyser II (Qiagen) (30Hz for 2mins) Single cell libraries were contructed using 10X Genomics Chromium Single-Cell 3' Gel Bead and Library V2 Kit, suspending cells in PBS with 5% FBS to a final concentration of 1 x 10^6 cells/ml (1000 cells per ul) and loaded in each channel with a target output of 3000 cells. Reactions performed with Bio-Rad C100 Touch Thermal Cycler with a 96 Deep Well Reaction Module, 12 cycles for cDNA amplificationand OCR indexing. Whole spleen RNA libraries were contructed using Illumina TruSeq Stranded Total RNA kit paired with the Ribo-Zero rRNA removal kit