title: NextSeq 500 paired end sequencing; RNAseq of follicular helper T cell from Elp3 deficient mice after Complete Freund Adjuvant immunisation
design: RNAseq of follicular helper T cell from Elp3 deficient mice after Complete Freund Adjuvant immunisation
library_name: WT3_p
library_construction_protocol: Mice received in both hind legs via subcutaneous injection 50 µg ovalbumin (Sigma-Aldrich) emulsified 1:1 with Complete Freund Adjuvant (Sigma-Aldrich). On D8 post-vaccination the inguinal lymph nodes were extracted and 90000 alive CD19- CD4+ GITR- CD44+ CXCR5+ PD1+ cells (TFH) were sorted in 1mL Trizol 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). RNAs were quantitatively and qualitatively verified using Agilent RNA 6000 Pico Kit run on an Agilent 2100 Bioanalyzer.  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