title: NextSeq 500 paired end sequencing; RNAseq of naive CD4 T cells deficient for Elp3 activated for 18h against WT control
design: RNAseq of naive CD4 T cells deficient for Elp3 activated for 18h against WT control
library_name: WT1_p
library_construction_protocol: Naïve CD4 lymphocytes were purified from spleen and lymph nodes with Naive CD4+ T Cell Isolation Kit (Miltenyi Biotec)). 2x10^5 naïve CD4 lymphocytes were activated in an anti-CD3e coated plate in complete RPMI 1640 medium with free anti-CD28. After 18h of culture, the cells were stained with a CD4 antibody and a viability dye and 90000 alive CD4 T cells were sorted into 1mL Trizol per sample with a FACS Aria 2. Total RNA was purified using Direct-zol® kit (Zymo Research) following manufacturer's recommendations. Eluted RNAs were incubated with DNase for 10 min at room temperature in a total volume of 200 µl using the RNase-Free DNase Set from Qiagen, followed by purification on RNeasy® Micro columns (Qiagen). cDNA was synthesized with 10 ng of total RNA using the SMART-SeqTM v4 UltraTM Low input RNA Kit for Sequencing (Clontech Laboratories) and amplified by 7 cycles of LD PCR. The sequencing library was generated using the Nextera XT DNA Library Preparation (Illumina) according to manufacturer's instructions. Libraries were submitted to a quality control on a QIAxcel (Qiagen) and to quantification using a KAPA Library Quantification Kit (Kapa Biosystems).
attributes.tag: Experimental Factor: genotype
attributes.value: wild type genotype