title: GSM8823458: E13_5_retina_Isl1_Rep1; Mus musculus; OTHER
library_construction_protocol: E13.5 retinae were isolated and nuclei extracted immediately by incubating in NE Buffer for 10 min on ice. The nuclei were incubated with activated concanavalin A-coated magnetic beads at room temperature for 10 min. The nuclei-beads were collected using magnetic stand and resuspended in 50 μl ice cold antibody buffer. 0.5 μg antibody against ISL1 (rabbit, AB4326, Millipore; rabbit, ab20670, Abcam) was added to each sample and mixed on a rotator at 4°C overnight or at room temperature for 2 h. The antibody-bound nuclei-beads were isolated and resuspended in 50 μl of ice cold Digitonin 150 Buffer. 0.5 μg of anti-rabbit IgG secondary antibody (EpiCypher #13-0047 and #13-0048) was added and samples were incubated for 30 min at room temperature on a rotator. After two washes in ice cold Digitonin 150 Buffer, nuclei-beads were resuspended in 50 μl of ice cold Digitonin 300 Buffer, 2.5 μl of CUTANA pAG-Tn5 solution (EpiCypher #15-1017) was added, and nuclei-beads were incubated for 1 h at room temperature on a rotator. The samples were again washed twice in ice cold Digitonin 300 Buffer, resuspended in 125 μl of cold Tagmentation Buffer, and incubated at 37°C for 1 h. Then, 2.5 μl of 0.5 M EDTA, 1.25 μl of 10% SDS and 1.1 μl of 20 mg/ml Proteinase K were added to each sample and samples were incubated at 70°C for 10 min or 55°C for 30 min. After this, DNA fragments were purified and collected in 30 μl of ddH2O using Cycle Pure Kit (Omega, D6492-02). 21 μl of DNA fragments were transferred to a PCR tube, to which 2 μl of barcoded i5 and i7 primers and 25 μl CUTANA High Fidelity 2x PCR Master Mix (EpiCypher, #15-1018) were added. PCR was performed using the following conditions: 72°C for 5 min, 98°C for 30 s, and 20 cycles of 10 s at 98°C and 10 s at 63°C, followed by an extra 1 min extension at 72°C. The PCR product was analyzed on an Agilent Fragment Analyzer. Combined DNA libraries were cleaned up using AMPure XP beads (#A63880, Beckman Coulter). Each pooled DNA library of 10 samples was resuspended in 40–80 μl water and quantified using a NanoDrop (Thermo FIsher Scientific). Library fragments were sent for sequencing commercially at Azenta Life Sciences. Sequencing was performed on Hiseq 4000 (Illumina) with 2 x 150 paired-end reads and a depth of 10 million reads per sample.