GSM8679456: E15.5 mouse metacarpal, digit 3, rep 3, MiSeq (RNAseq); Mus musculus; RNA-Seq
Embryos were microdissected under a light microscope in 10% fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) on ice and anatomically sexed. The pooled phalanges (proximal and distal for digit I; proximal, intermediate, and distal for digits III and V) and metapodials were collected separately for digits I, III, and V for both the fore- and hind limbs. The right and left sides of each element were pooled together. Each element was stripped of soft tissue and collected in a 2-ml tube containing 200 ul of TRIzol  and one 5-mm stainless steel bead. Each sample was then homogenized at 50 Hz for 2 min, followed by 1 min on ice, and a second homogenization at 50 Hz for 2 min. Samples were stored at -80˚C until RNA extraction was performed.  For each RNA extraction, samples were incubated at room temperature for 5 min and then centrifuged at 4˚C for 5 minutes at 12,000 x g. The supernatant was collected and transferred to a clean tube, to which 200 ul of chloroform was added per 1 ml of TRIzol. Each sample was then vortexed briefly and incubated at room temperature for 2 minutes before being transferred to a MaXtract tube  and centrifuged at 4˚C for 5 minutes at 12,000 x g. Following centrifugation, the aqueous phase was removed and transferred to a new microcentrifuge Eppendorf tube and an equal volume of 100% ethanol was added. After completion of this phenol-chloroform step, the RNA extraction continued using the Direct-zol RNA MicroPrep kit  following the manufacturer's protocols and eluted in 15 ul of nuclease-free water. Samples were stored at -80˚C until library preparation. Aliquots of the samples were nanodropped to quantify the extracted RNA and to determine 260/230 and 260/280 values and then run on a HS RNA Tapestation tape to their determined their RNA integrity number (RIN) value. Only samples with RIN scores higher than 6 were used for subsequent steps so long as the average RIN for all tissues from that sample was greater than 7. Samples were then sent out for 3 rounds of sequencing on an Illumina MiSeq/NovaSeq 6000 at the Harvard Bauer Core in order to obtain ~40 million reads per sample. Takara SMART-Seq v4 Ultra Low Input RNA Kit Library Preparation (2 samples also prepared using Kapa mRNA HyperPrep kit for comparison with human samples)