GSM8080115: Replica 3 0615 SCR; Homo sapiens; RNA-Seq
For RNA extraction from cells, J-Lat 6.3 cells were stimulated with P/I and sorted based on their GFP expression (n=4). For analysis of transcriptome upon Cytor depletion in primary CD4+ T cells, cells were stimulated with CD3/CD28 beads and then subjected to Cytor KD by transducing cells with lentivirus that drive the expression of shRNA that target Cytor (n=3). RNA from both cell types was purified utilizing the RNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. The integrity of the isolated RNA was tested using the Agilent High Sensitivity RNA Kit and Tapestation 4200 Libraries construction for  sequencing was performed using the NEBNext Ultra II RNA kit (NEB;E7770L). Quantification of the library was performed using dsDNA HS Assay Kit and Qubit 2.0 (Molecular Probes, Life Technologies), and qualification was done using the Agilent D1000 Tapestation Kit and Tapestation 4200. 150 nM of each library was pooled together and was diluted to 4nM according to NextSeq manufacturer's instructions. 1.6 pM was loaded onto the Flow Cell with 1% PhiX library control. Libraries were sequenced on an Illumina NextSeq 500 instrument, 75 cycles of single-read sequencing.