Illumina HiSeq 2500 paired end sequencing; The TAB1-p38α complex aggravates myocardial injury and can be targeted by small molecules
The TAB1-p38α complex aggravates myocardial injury and can be targeted by small molecules
Left ventricle was harvested from mouse. TAB1 (V390A, Y392A, V408G, and M409A) KI mouse generation: The targeting vector 5′ homology arm and 3′ homology arm was amplified from BAC DNA and confirmed by end sequencing; the mutations V390A, Y392A, V408G, and M409A were introduced into exon 10 by site-directed mutagenesis with QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). The targeting construct was electroporated into C57BL/6 ES cells, 375 G418-resistant colonies were picked, and the PCR assays were performed with primers 5PCR_F and 5PCR_R. Thirty-four potential targeted clones were identified. Six of them were expanded and frozen. The Southern blot analysis was conducted with 5′-probe, 3′-probe, and Neoprobe. The genomic DNA of the potential clones were digested with Bg1II for the 5′-probe and analysed by Southern blot for a 8.0-Kb band from the WT allele and a 10.0 Kb band for the recombinant allele; for the 3′ probe, the genomic DNA was digested with XmaI and analyzed by Southern blot for a 7.8-Kb band from the WT allele and a 5.7-Kb band for the recombinant allele. Regarding the Neo probe, the genomic DNA was digested with NsiI and analyzed by Southern blot for a 6.6-Kb band for the recombinant allele. TAB1 (V390A, Y392A, V408G, and M409A) F1 mice were generated from the ES cell lines 2C9. Five (3 males and 2 females) out of 8 pups were identified as positive by PRC screening: the expected 261-bp fragment from WT allele and 405-bp fragment from recombinant allele were represented as positive F1 mice. The primers used for the screening were: Neo_Del_F and Neo_Del_R. The primers sequences used were: 5PCR_F: 5′-GCGGAGTATTAGGAGCCTGAGGGT-3′, 5PCR_R: 5′-GCTGACCGCTTCCTCGTGCTTTA-3′, 5′-probe_F: 5′-CAGCCACTTGTGAGACCAGAGGA-3′, 5′-probe_R: 5′-GGACAAGCCTGTGAGGTGATGACA-3′, 3′-probe_F: 5′-GGTCCCTCACAAGGGTTAAGCAACT- 3′, 3′-probe_R: 5′-GAACTTGTCTGTGCTCTGAGCTGGC-3′, Neo-probe_F: 5′-CCTGAATGAACTGCAGGACGAGG- 3′, Neo-probe_R: 5′-AGCTCTTCAGCAATATCACGGGTAGC-3′, Neo_Del_F: 5′-GCTGGCCTTGCTCAACTCCAG-3′, Neo_Del_R: 5′-GACCATCTGTCTCATACCTGACCTCAC- 3′ RNA was extracted from 40 – 60mg of mouse left ventricle using 900ul of TRI-Reagent® (Ambion) followed by MagMax™-96 for Microarrays Kit (Ambion, AM1839) according to the Spin Procedure. In summary, tissues were homogenised in TRI- Reagent® for 5 – 10 minutes at room temperature using TissueLyser (QIAGEN). The homogenate was mixed with 0.1 volumes of 1–bromo–3–chloropropane (BCP) and centrifuged at 12 000 x g at 4°C for 10 minutes. The 100ul of aqueous phase was collected and mixed with 50ul of 100% isopropanol. RNA was bound to RNA Binding Beads. The Beads were washed twice with wash solution, and total RNA was eluted in Elution buffer. RNA was quantified using the NanoDrop 1000 spectrophotometer (Thermo Scientific). RNA integrity was analysed using the the Bioanalyzer RNA Nano Kit (Agilent).  100 ng of intact high quality total RNA (RIN>9) from each sample was used as input to generate libraries for RNA-seq using the NEBNext Ultra Directional kit (NEB, Cat.no: E7420) following the manufacturer's recommendations. This protocol involved an initial step of rRNA depletion, followed by fragmentation prior to first cDNA synthesis. Each sample was barcoded during second-strand cDNA synthesis with indices for illumina sequencing. A single index was used per sample (illumina indices 1 to 10). Libraries were enriched by PCR for 15 cycles.  The resulting libraries were purified using Agencourt AMpure Xp beads (Beckman). Library quality was assessed on the Bioanlyzer 2100 using DNA High Sensitivity kit. The library size distribution was determined using Agilent 2100 expert software.
King's College London
E-MTAB-6460:Sample 1_p