GSM4891339: PDE4_ProxE12.5limb_replicate1; Mus musculus; OTHER
E12.5 wild-type proximal forelimb segments were dissected in cold 10% fetal bovine Serum (FCS) in PBS. Dissected proximal forelimbs were pooled (10-13 pairs) and dissociated with collagenase (Sigma-Aldrich) and filtered through a 35 micron mesh to isolat Nuclei were digested with NlaIII (New England Biolabs) and ligated with T4 DNA ligase HC (Promega) under dilute conditions to promote intramolecular ligation. Samples were then digested with DpnII (New England Biolabs) and again ligated with T4 DNA ligase HC (Promega) under dilute conditions. Templates were amplified with inverse PCR primers containing Illumina adapters using Expand long template polymerase (Roche). The sequences of primers for each viewpoint were (illumina adapters underlined):  Shox2 Forward/Reading primer: 5'-AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTCCAATTAAGAAAATATGTGGCATG; Reverse Primer: CAAGCAGAAGACGGCATACGAAGAATGTGAAGTTTGGTCCC; LHB-A: Forward/Reading primer: AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGCTTAGTAAAGACTAATTATTCATG; Reverse Primer: CAAGCAGAAGACGGCATACGAAATGACATTATTATAAAATGCAATACTCT; PDE4: Forward/Reading primer:   AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCTGATTCTCCTGCATG  Reverse Primer: CAAGCAGAAGACGGCATACGAAGTTATAAAGATGATTAAGCTCTGATC  Libraries were sequenced together for each replicate on the Illumina HiSeq 2500 following the manufacturer's protocols. Viewpoint-specific primer sequences were used for subsequent demultiplexing.