GSM2691378: D3 Egr1-/- E24 OS Close; Mus musculus; RNA-Seq
Eyes were enucleated, put into eppendorf tubes filled with RNAlater (Thermo Fisher), and total RNA extracted using the RNeasy Mini Kit (Qiagen). Poly(A)+ RNA was selected twice from total RNA using Dynal oligo magnetic beads (Invitrogen). Double-stranded cDNA was synthesized using SuperScript (Invitrogen) and fragmented using Bioraptor (Diagenode). Adaptors (Illumina) were ligated to the dsDNA using T4 DNA ligase (New England Biolabs) after end-repair using End-It kit (Epicentre) and dA addition. Libraries were size-selected for 250-400 bp fragments, purified using 2% E-Gel (Invitrogen), and amplified for 18 cycles by PCR with PE 1.0 and PE 2.0 primers (Illumina) and Phusion High Fidelity PCR Master Mix. PCR products were barcoded (indexed) and sequenced on Illumina HiSeq 2000 platform.