GSM8096868: E9.5 posterior trunk, Prtg-/-, sample 2; Mus musculus; RNA-Seq
RNA was extracted using Tissue Total RNA Mini Kit (Geneaid). 1 μg total RNA was used for following library preparation. Next generation sequencing library preparation was constructed according to the manufacturer's protocol (NEBNext® Ultra™ RNA Library Prep Kit for Illumina®). The poly(A) mRNA isolation was performed using a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). The mRNA fragmentation and priming was performed using NEBNext RNA First Strand Synthesis Reaction Buffer and NEBNext Random Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified double-stranded cDNA obtained by AxyPrep Mag PCR Clean-up (Axygen) was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tail; this was done in a single reaction, and was followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~420 bp (with the approximate insert size being 300 bp) were recovered. Each sample was then amplified by PCR for 13 cycles using the P5 and P7 primers, with both primers carrying sequences that are able to anneal with the flow cell in order to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using a Qsep100 (Bioptic), and quantified by Qubit 3.0 Fluorometer (Invitrogen).