GSM5583206: Brn3b_RNAseq_Rep1; Mus musculus; RNA-Seq
Retinas from embryos at E16.5 were dissected in HBSS buffer without Ca 2+ and Mg 2+ . In the case of retinas from Pou4f2 SunGFP embryos the ventrotemporal region was remove by dissection. Retinas were digested and disaggregated with the help of a P1000 using an enzymatic solution composed by colagenase, trypsin 23/08/2138 0,125%, BSA 0.2 mg/mL and DNAse 1μl/mL during 25' at 37ºC. Digestion was stopped by adding DMEM with fetal bovine serum at 10% and disaggregation of the tissue was checked under microscope. The suspension was filtered and centrifugated at 1,250 rpm during 5' at RT. The pellet was resuspended and sorted by flow cytometry to harvest our interest cells. RNA was extracted from sorted cells using PicoPure TM RNA Isolation Kit (Arcturus) and libraries were made and sequenced in a HiSeq 2500 sequencer (Illumina, Inc). Retinas from embryos at E16.5 were dissected in HBSS buffer without Ca 2+ and Mg 2+ . In the case of retinas from Pou4f2 SunGFP embryos the ventrotemporal region was remove by dissection. Retinas were digested and disaggregated with the help of a P1000 using an enzymatic solution composed by Colagenase, Trypsin 0,125%, BSA 0.2 mg/mL and DNAse 1μl/mL during 25 min at 37ºC. Digestion was stopped by adding DMEM with fetal bovine serum at 10% and disaggregation of the tissue was checked under microscope. The suspension was filtered and centrifuged at 1250 rpm during 5 min at RT. The pellet was resuspended and sorted by flow cytometry to harvest 50000 green fluorescent cells. Cell suspension was spin down at 500 g for 5 min at 4ºC and treated with cold lysis buffer (10mMTrisCl ph7.4, 10mM NaCl, 3mM MgCl 2 , 0.1% Igepal). The suspension was centrifuged at 500 g during 10 min at 4ºC. We discard the supernatant, and we make sure that the pellet was placed on ice. After that we continued with the transposition reaction and purification following the protocol as (Buenrostro et al., 2013, 2015) . Libraries were purified and sequenced by an Illumina HiSeq 2500 sequencer (Illumina, Inc).