title: GSM7699141: LIHDIIP03 at 12W; Homo sapiens; RNA-Seq
library_construction_protocol: Following the PBMC isolation from fresh blood of different participants at various time points/visits, CD4+ T cells were first enriched with human CD4 microbeads (130-045-101, Miltenyl Biotec) following the manufacture's recommendations while the remaining fractions of PBMC were immediately cryopreserved as described in other sections of the manuscript. The CD4+ T cells were then immediately shipped to our sorting flow-cytometry facility at 4°C. The CD4+ T cells were first stained in Brilliant Stain Buffer (563794, BD) using all the Abs with specified dilution factors listed in Table S4 (of the manuscript). Incubate for 30 min at 4°C in the dark, briefly vortex once after 15 min. Wash the cells twice: centrifuge at 350g, 10 min, 4°C. Re-suspend the cells at a concentration of 10 million per ml. Proceed to aseptic sorting 20 PSI, with 100 um nozzle and Purity mode in a BD Aria III sorter (for the FACS gating strategy, please refer to Figure S6A of the manuscript). After sorting, cells were centrifuged at 350g, 10 min, 4°C, pelleted and lysated in 700 ul of the Qiazol buffer (Qiagen). Transfer the samples into 1.5 ml Eppendorf tubes and immediately freeze at -80°C until RNA extraction. RNA was extracted using RNeasy Micro kit (Qiagen) following the manufacture's recommendations. RNA-seq was performed at the EMBL Genomics Core Facility (Heidelberg, Germany) using single-read 75bp by NextSeq 500 with ~ 50 Million reads/sample. The NEBNext Ultra II RNA Library Prep Kit was used and the rRNA depletion step was included.