title: Illumina MiSeq paired end sequencing; Sepsis induces long-lasting impairments in T helper cell responses despite rapid numerical recovery of T-lymphocyte populations
design: Sepsis induces long-lasting impairments in T helper cell responses despite rapid numerical recovery of T-lymphocyte populations
library_construction_protocol: Mice were sacrificed at three different days after sepsis. Spleens were harvested and total RNA was extracted using Trizol Reagent from 10*7 cells. mRNA was purified with Directzol RNA MiniPrep. After synthesis complementary DNA (Primer was T cell specific: C1 segment of β-chain) MultiPlex PCR was performed (amplified with primers spanning the whole complementarity-determining region (CDR)3 starting from 5´ Vβ 1,2,3,5,9,12-2,13-1,13-2,13-3,14,16,17,19,20,26 and from 3´ with the C1 segment of the β-chain) and finally purified with agarose gel electrophoresis and a Gel DNA-Recovery Kit. This method ensured that only TCR specific DNA was sequenced. We did not differentiated the T cell subpopulations before extracting RNA. Total RNA was extracted using Trizol Reagent (Ambiom) from 10^7 unsorted spleen cells of every septic and sham mouse. mRNA was purified with Direct-zol RNA MiniPrep (ZymoResearch). Complementary (c)DNA synthesis was performed with M-MLV reverse Transcriptase (Affymetrix), RNasin Plus (Promega) and reverse transcriptase Primer (C1 segment of β-chain, Biotez). cDNA was amplified with primers spanning the whole complementarity-determining region (CDR)3 regions starting from 5´ Vβ 1,2,3,5,9,12-2,13-1,13-2,13-3,14,16,17,19,20,26 and from 3´ with the C1 segment of the β-chain. ultiplex PCR was performed using a Multiplex PCR Kit (Qiagen) and finally purified with agarose gel electrophoresis and a Gel DNA-Recovery Kit (Zymoresearch).
attributes.tag: Experimental Factor: disease
attributes.value: sepsis
attributes.tag: Experimental Factor: sampling time point
attributes.value: 8