GSM3611600: Day 2; Mus musculus; RNA-Seq
Total RNA of purified donor fibroblasts was isolated by using RNeasy mini kit (Qiagen). Whole transcriptome of purified transferred cells was amplified and acquired by SAGE as described previously with minor differences (GSE42564 and GSE110533). Purified total RNA was dissolved in lysis buffer (1% LiDS, 100 mM Tris-HCl pH 7.5, 500 mM LiCl, 10 mM EDTA, 5 mM DTT) and mRNA was trapped on Dynabeads M270 streptavidin (Thermo Fisher Scientific) labeled with biotinylated oligo-dT-containing DNA adapters as described previously (GSE110533). On-beads reverse transcription and second strand synthesis was performed by adding RT mix (1x First-strand buffer, 0.5 mM dNTP, 2.5 µM DTT, 1U/µl RNaseIn Plus (Promega),  1.2 µM SMART-LNA oligo (Exiqon), 20U/µl Superscript II (Thermo Fisher)) and incubating 37ºC for 1 hour, 70ºC for 10 min, and hold at 4 ºC. Then, whole transcriptome was amplified using PrimeSTAR DNA polymerase (Takara) by performing below program; 98 ºC 2min, 18 cycles of [98 ºC 20 sec, 60 ºC 15 sec, 68 ºC 4 min], 68 ºC 4 min, and hold at 4 ºC. SAGE libraries were constructed by using each amplified whole transcripts as described previously (GSE42564). 3'SAGE
GEO Accession
GSM3611600