GSM5589164: E14.5 mouse embryonic maxillary prominence_rep2; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol from freshly dissected maxillary prominence tissues from 7 littermates. For each developmental stage (E14.5), three independent RNA samples were prepared. RNA purity was checked using the kaiaoK5500®Spectrophotometer (Kaiao, Beijing, China). RNA integrity and concentration were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). For each sample, 2 μg total RNA was used as input material for the library preparations. Sequencing libraries were then generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and RNase H. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. The library fragments were purified with QiaQuick PCR kits and eluted with EB buffer, followed by terminal repair, A-tailing and adapter ligation. The ligated products were retrieved and PCR amplification was performed to generate sequencing libraries. RNA concentration of library was measured using Qubit® RNA Assay Kit in Qubit® 3.0 to preliminary quantify and then dilute to 1ng/μl. Insert size was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA), and qualified insert size was accurately quantified using a StepOnePlusTM Real-Time PCR System. The clustering of the index-coded samples was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the libraries were sequenced on an Illumina HiSeq X ten platform and 150 bp paired-end reads were generated.