GSM5017911: RNA-seq FL bud E11.5 replicate 3; Mus musculus; RNA-Seq
Per biological triplicates (n=3), a pair of mouse forelimb buds at E9.75 (28-30 somites), E10.5 (34-36 somites) and E11.5 (43-45 somites) stages and chicken wing buds at HH20, HH22 and HH24 stages and were microdissected in cold PBS. These samples were transferred to RNA later (SigmaR0901) after an overnight incubation at 4°C and samples were stored at -20°C. Total RNA was extracted using the RNeasy micro kit (Qiagen Ref.74004). Homogenization of tissue in RLT buffer was done by sheering the sample using a 25G needle for 20 times. An on column DNA digestion (Qiagen Ref.79254) was done during the extraction to minimize the gDNA contamination. RNA integrity was checked on a Agilent Fragment Analyser (RNA analysis kit DNF-472-0500). The RNA concentration was measured by QubitTM RNA HS Assay kit (Ref Q32855). 500ng total RNA was used to prepare RNA-seq libraries using NEB non directional NEBNext® Ultra™ II RNA Library Prep (E7775), using the polyA mRNA workflow and 9 cycles of PCR amplification. Barcoded poly-A RNA-seq libraries were pooled equal molar and sequenced on the Illumina Nextseq500.