B cells from lncCSR IgA WT/WT mouse spleen for ChIP-seq (anti-CTCF)
Resting B cells derived from spleen were crosslinked with formaldehyde 37% (Sigma Aldrich F8775-500 ml) for 10 minutes with gentle inversion/rotation before the addition of 100 ul/ml of 1.375M glycine to quench the reaction for another 10 minutes. Cells were washed several times with PBS before being suspended in SDS lysis buffer with protease inhibitors (1% SDS, 10 mM EDTA, 50 mM Tris plus 1 complete mini EDTA-free protease inhibitor solution tablet/10 ml lysis buffer). Sonication was performed on ice using a Branson Sonifier 250 apparatus for 25 cycles, each cycle comprising 20 s of sonication at duty cycle 30% followed by a 2 min rest period. The cell lysate was centrifuged at maximum speed for 10 minutes at 4C and 2% of the material was snap-frozen as input.  Lysates were pre-cleared with protein A/G plus agarose solution and ChIP-grade rabbit IgG for 1 h. Immunoprecipitation of lysates was performed overnight at 4 C with gentle rotation using Anti CTCF antibody (Abcam cat no. ab70303). Sonicated herring sperm DNA (Promega D1815) and protein A/G plus agarose solution were added for 90 min with continued rotation. Subsequently the beads were washed by the standard series of washes (low salt, high salt, LiCl, and TE) and ChIP products were eluted followed by RNaseA treatment overnight at 60C and proteinase K treatment for 12 h at 37C followed by 2h at 55C. ChIP DNA was purified by phenol-chloroform-isoamyl alcohol addition and recovered using phase-lock gel heavy tubes and then precipitated using ethanol. The final concentration of DNA was measured using the Qubit 3.0, followed by library preparation using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB cat no E7645S and cat no. E7600S).