GSM5931424: Total RNA-seq Hindlimb (Pbx1/Pbx2 ctrl; rep. 2); Mus musculus; RNA-Seq
Hindlimb buds from mutant and littermate controls were dissected individually at the indicated gestational days and snap frozen in dry ice. After genotyping of individual samples, each biological replicate consisted of one individual mutant or littermate embryo, with a total of 3 biological replicates per genotype. RNA was extracted using the RNeasy Plus Micro kit (Qiagen, #74034) and RNA quantification was performed using the Qubit RNA HS Assay Kit (Invitrogen, #Q32852). Quality control of input RNA was performed using the RNA 6000 Pico kit (Agilent, #5067–1513) on a 2100 Bioanalyzer (Agilent). All RNA samples used for library preparation had RIN>9. RNA sequencing libraries were prepared from 100 ng of input RNA using the non-directional kit NEBNext Ultra™ II RNA Library Prep Kit for Illumina (NEB, #E7775) with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, #E7490) to capture polyA RNAs. Library size and quality was checked using an Agilent 2100 Bioanalyzer with the High Sensitivity DNA kit (Agilent, #5067–4626), and library concentration was determined with the QuBit dsDNA HS Assay kit (Invitrogen, #Q32854). Libraries were normalized to 10 nM, pooled, and sequenced in an Illumina HiSeq 4000 to generate 50 base pair single-end reads.