title: GSM6032004: D28_TFL_Mouse4_Run3 [S15-Day27-sort3-Tfl-Mouse4]; Mus musculus; RNA-Seq
library_construction_protocol: OT-1 enriched T-cell populations were obtained through anti-CD45.1 biotin anti-biotin microbead staining and magnetic cell separation (Miltenyi Biotech). Highly purified populations (>95%) of the indicated phenotypes were obtained through flow cytometry-based cell sorting (FACSAria Fusion cell sorter). RNA was extracted using the Agencourt RNAdvance Cell v2 kit (A47942, Beckman Coulter) and RNA integrity number (RIN) and yield were assessed using RNA 6000 Pico Kit (5067-1513, Agilent). Only samples with RIN>8 were used for downstream cDNA synthesis and library preparation. cDNA synthesis and PCR amplification using 1 ng of total RNA from each sample (see "Design_Combined_Run2+3+17+19.txt") was performed using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (634891, Takara/Clontech). After cDNA synthesis, each sample was subjected to 12 cycles of PCR amplification. The concentration of generated amplicons was determined using the Agilent High Sensitivity DNA Kit (5067-4626, Agilent). 150 pg of the resulting amplified cDNA was used for library preparation with the Illumina Nextera XT DNA Library reagents (FCSHAPING 131-1024, Illumina). After PCR amplification of the fragmented libraries, the samples were purified with (0.6x) Agencourt AMPure XP beads and eluted in 10 μl of molecular grade water. The quality of the resulting library was assessed using the High Sensitivity DNA Kit (5067-4626, Agilent). The library quantification was performed based on the Illumina recommendations (SY-930-1010, Illumina), with the use of KAPA SYBR FAST qPCR Master Mix (KK4600, Kapa Biosystems).