Escherichia coli ligase

Total RNA from ESCs, EBs, 293T cells, MBs, and MTs was isolated with RNeasy (QIAGEN) and reverse transcribed using SuperScript III and random hexamers (Life Technologies) to synthesize the first strand. Second strand was synthesized with deoxyuridine triphosphate to generate strand asymmetry using DNA polymerase I [M0209L, New England Biolabs (NEB)] and the Escherichia coli Ligase (L6090L, Enzymatics). RNA-seq libraries were constructed using the protocol described in (43 (link)).

Total RNA extractions were performed using the Roche High pure RNA isolation kit. Superscript III reverse transcription reagents (Invitrogen) and random hexamers were used to prepare cDNAs. For qPCR quantification, 5 μl of SYBR Green I Master mix (Roche), ROX reference dye, 1 μl of IDT PrimeTime Primer set for corresponding assays, and 1 μl of cDNA were mixed for PCR amplification and detected by QuantStudio 5 real-time PCR systems instrument. For quantitative analysis, Hoxa9, Hoxb4, Hoxb13, Gata2, Gata6, Pou5f1, and Ccna2 expression was normalized to Actb expression. Primers were ordered from Primetime IDT. For RNA-seq, the first strand was synthesized using reverse transcription via Superscript III and random hexamers. The second strand was synthesized with deoxyuridine triphosphate to generate strand asymmetry using DNA polymerase I (NEB, M0209L) and the Escherichia coli ligase (Enzymatics, L6090L). RNA-seq libraries were constructed using the protocol described in (1 (link)), quantified by Qubit double-stranded DNA HS Assay Kit quality, and checked by High Sensitivity D1000 ScreenTape. Libraries were then sequenced as 50-bp single-end reads on a NovaSeq 6000 platform.