Truseq stranded mrna ht kit

Illumina sequencing libraries were generated with the TruSeq Stranded mRNA HT kit (Illumina) using an Eppindorf ePMotion 5075 robot and were sequenced to a depth 45–60 million reads per sample on Hi-Seq 2500 instruments at the Roy J. Carver Biotechnology Center at the University of Illinois. All sequencing data generated in this study have been deposited to the GEO database under Accession number GSE184549. Pairwise comparisons of E18, P1 and P3 samples were completed as previously described in detail [37 (link)], with results provided in S2 Table. For functional analysis, genes that were found to be differentially expressed at fdr < 0.05 were first filtered for absolute fold change >1.5, and uploaded to the ToppCluster web analysis tool [61 (link)] using default conditions (Bonferroni correction, fdr <0.05). Selected categories are summarized in Fig 2, with full ToppCluster Results reported in S3 Table.

Samples were ground with liquid nitrogen, and RNA purification was performed by the Tri-Reagent method (Sigma-Aldrich, San Louis, MO, USA). Libraries were constructed with the TruSeq Stranded mRNA HT kit (Illumina, San Diego, CA, USA), and sequencing was performed by the Mass Sequencing Unit of the Biotechnology Institute (UNAM, Cuernavaca, Mor, México) with a NextSeq 500 Sequencer (Illumina, San Diego, CA, USA) using 150 cycles of pair-end readings.

Total RNA was extracted using an RNeasy mini kit (Qiagen) according to the manufacturer’s protocol. The RNA concentration was measured using NanoDrop (Thermo Fisher Scientific), and the quality of RNA was analyzed using the Agilent 4200 TapeStation (Agilent, Santa Clara, CA, USA), RNA Screen Tape (Agilent), and RNA reagent kit (Agilent), after which the samples were stored at −80 °C. The method of the confirmation of RNA quality was in accordance with the manufacturer’s instructions. The RNA was used to prepare a cDNA library for RNA sequencing. TruSeq Stranded mRNA HT kit (Illumina, San Diego, CA, USA) was used for library preparation. The concentration of the prepared library was measured by Qubit (Thermo Fisher Scientific). The Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) was used, and the method was performed according to the kit manual. The length of the DNA fragment of the library was analyzed by the Agilent 4200 TapeStation. The DNA fragment length was determined using the D1000 ScreenTape (Agilent) and the D1000 reagent kit (Agilent). Each library was stored at −20 °C. The libraries with confirmed quality were sequenced with pair-end sequencing using the NextSeq 500 System (Illumina) and the NextSeq 500/550 High Output Kit v2 (150 Cycles) (Illumina). The library preprocessing was performed according to the manufacturer’s instructions.