GSM9008988: E11.5 WT replicate 1; Mus musculus; RNA-Seq
Epithelium and mesenchyme cells were sorted using FACS (Sony SH800S) as described earlier, collecting Epcam positive and negative cells directly into separate tubes containing RLT RNA extraction buffer. Post-genotyping of individual embryos, RNA from 4 embryos per group (Pbx1/2 mutant and control) was pooled. A total of four biological replicates were analyzed for each genotype. RNA extraction was performed using the RNeasy Plus Micro Kit (Qiagen, #74034), and RNA quantity was assessed using the Qubit RNA HD Assay Kit (Invitrogen, Q32852). RNA quality was checked using an RNA 6000 Pico kit (Agilent, Cat: 5067-1513) on a 2100 Bioanalyzer (Agilent), ensuring all samples for library preparation had an RIN greater than 9. PolyA RNAs were isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, #E7490). RNA sequencing libraries were prepared from 100ng of RNA using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina (NEB, #E7775). The library size and quality were verified on an Agilent 2100 Bioanalyzer using the High Sensitivity DNA kit (Agilent, Cat: 5067-4626), and library DNA concentrations were measured using the Qubit dsDNA HS Assay Kit (Invitrogen, Q32854). RNA libraries were prepared according to 10X protocol.