NextSeq 500 sequencing; RNA-Seq data of Trpm7+/+ and Trpm7-/- embryonic stem cell clones
RNA-Seq data of Trpm7+/+ and Trpm7-/- embryonic stem cell clones
Two-cell-stage (1.5-dpc) embryos were flushed from superovulated females (CD1, Janvier) with M2 (HEPES-buffered) Medium and electrofused in M2 medium supplemented with 0,3 M Mannitol and 0.3% BSA within 30 - 45 minutes after isolation, essentially as described(Kubiak and Tarkowski, 1985). The settings of the pulse stimulator (BLS, Hungary, Model CF-150/B) were as follows after optimization in zygotes of the CD-1 strain: # of pulses: 2,voltage: 36 V, pulse duration: 35 μs; alternating current (AC) for positioning of embryos: 1.4 V. Only fused embryos were cultured in KSOM medium (COSMO BIO CO., LTD.) for two days to the blastocyst (E3.5) stage. TRPM7 +/+ ES cells of clone 03 (passage 17) and Trpm7 −/− ES cells of clone 14 (passage 15) were microinjected in seven independent experiments into tetraploid blastocysts and transferred to the uterus of pseudo-pregnant CD1 foster mothers. Embryos were isolated at E10.5. RNA isolation was performed using the RNeasy Mini kit (Qiagen) according to manufacturer's recommendations for cells, including on-column DNase digest. 20 ng total RNA was used per sample to prepare genomic libraries (average fragment size of 300 bp) using Ovation® SoLo RNA-Systems Kit (Tecan 0407-32) as described in the protocol provided by the manufacturer.