GSM2813853: Erythroid cells fixed with 5% formaldehyde replicate 1; Mus musculus; OTHER
Spleens were gently dissociated into a single cell suspension and cells were passed through a 70 μm strainer to remove clumps. For ter-119+ cell selection, cells were stained with phycoerythrin conjugated anti-ter-119 antibodies and positively selected using anti-phycoerythrin MACS beads before fixation with formaldehyde. The Capture-C technologies combine 3C library preparation with oligonucleotide capture for the desired viewpoint restriction fragments. Detailed description follows. The protocol used for the generation of the 3C libraries was similar to other 3C-based methods. Briefly, cells were crosslinked with 2% formaldehyde (this was varied for the reduced crosslinking experiments), quenched with cold glycine, washed in phosphate buffered saline, resuspended in cold lysis buffer and snap frozen to -80. Cells were thawed on ice, washed in Milli-Q dH2O and Dounce homogenized on ice. Cells were then resuspended with 0.25% SDS and restriction enzyme buffer and incubated at 37 °C for 1h at 1400 rpm on a Thermomixer, followed by a further incubation of 1h after the addition of Triton X-100 (final concentration 1.67%). An overnight digestion was performed using DpnII (final concentration 500U/ml; 37 °C / 1400 rpm). The digested chromatin was ligated overnight using T4 DNA HC ligase (final concentration 10U/ml; 16 °C / 1400 rpm). The samples were de-crosslinked overnight with Proteinase K at 65 °C, followed by a 30 min incubation at 37 °C with RNAse. A Phenol/Chloroform extraction was performed, followed by an ethanol precipitation and wash. Digestion efficiencies were assessed by gel electrophoresis and RT-PCR, which showed digestion efficiencies higher than 80%. The DNA contents of the DpnII 3C libraries were quantitated using a Qubit fluorometer. Different strategies were used for the sequencing library preparation. For the low-input experiments, the LI Capture-C or Tag-Capture-C protocol was used, for which 50-200 ng of DNA was adapter ligated using a strategy based on sonication combined with the NEBNext Ultra DNA Library Prep kit, or the Illumina Nextera DNA Library Prep kit, respectively. For the reduced cross-linking experiments, the NG Capture-C protocol was used, for which 5 ug of DNA was adapter ligated using a strategy based on sonication combined with the NEBNext DNA Library Prep kit. The quality of the libraries was assessed using the Agilent ScreenTape system. Biotinylated capture oligonucleotides were designed to the ends of the viewpoint fragments. 700 ng-2 ug of the adapter ligated libraries was hybridized with the biotinylated capture oligonucleotides, using the Nimblegen SeqCap reagents and an adapted protocol. The quality of the resulting captured library was assessed using the Agilent ScreenTape system. The libraries were sequenced using the Illumina MiSeq (150bp paired end reads) platform.
GEO Accession
GSM2813853