Neb next ultra library preparation kit

hMeDIP was performed using 5 µg of genomic DNA extracted from a population of 1.5 to 2 *10⁶ leptotene/zygotene spermatocytes (95% pure) (see above). Genomic DNA was obtained by phenol/chloroform extraction and then sonicated to a size of ~150 bp with a Bioruptor pico apparatus (Diagenode, B01060010). Then, Illumina adaptors were added using the NEB Next Ultra Library Preparation Kit (NEB7370S), without the final PCR step. Finally, hMeDIP was performed with the Active Motif hMeDIP Kit (AM, 55010), according to the manufacturers’ manual. Enriched fragments were then amplified by PCR using 12 cycles, as recommended by the NEB Next Ultra Library Preparation Kit. Sequencing was performed on a HiSeqX (2 × 150 bp).

Cells were cross-linked with 1% methanol-free formaldehyde for 10 min. After quenching with glycine, cells were washed three times with PBS. The cell pellet was treated with 40 U MNase for 5 min at 37°C and then stopped with 10× Covaris buffer (Covaris), and chromatin was sheared for 15 min with the Covaris S2 device (burst 200; cycle 20%; intensity 8). Immunoprecipitation was performed for approximately 5 × 10⁶ cells with anti-H3 antibody (Abcam ab1791, lot GR103864-1). Then chromatin was treated with RNase A and Proteinase K. Purified DNA was cloned into Illumina libraries with the NEBNext ultra library preparation kit (NEB). Paired-end reads were sequenced using Illumina HiSeq 2000.
RNA-seq was performed using total RNA extracted using a DNA-Free RNA kit (Zymo Research) as detailed in the Supplemental Methods. Libraries were prepared from RNA of WT and DKO ESCs using the TruSeq RNA sample preparation kit v2 (Illumina), clustered on cBot (Illumina) using TruSeq SR Cluster Kit v3 and sequenced by single-read 50-bp mode on a HiSeq 2000 v3 platform according to Illumina's instructions. RNA-seq analysis was performed in Genomatix (Genomatix GmbH) as detailed in the Supplemental Methods.

Patient‐derived cells were cross‐linked with 1% methanol‐free formaldehyde for 10 min. After quenching with glycine, cells were washed three times with PBS and the cell pellet was frozen in liquid nitrogen. For analysis, the cell pellet was thawed and treated with four units MNase per 1 × 10⁶ cells for 15 min. MNase was stopped with 10× covaris buffer, and the chromatin was sheared for an additional 15 min with the S2 covaris device. The soluble chromatin was then recovered and subjected to a background‐minimizing pre‐clearing step with an unspecific IgG antibody. For each ChIP assay, an equivalent of 3 × 10⁶ cells was used. After the IP, chromatin was digested with RNaseA and proteinase K. From the purified DNA sequencing, libraries were generated with the NEBNext Ultra library preparation kit (NEB). ChIP‐seq of both CTCF and EBF1 was done with the SimpleChIP‐seq kit from Cell Signaling Technology according to the manufacturer's instructions. After purification of the DNA, libraries were cloned with the NEBNext Ultra II library kit (NEB). The antibodies used for ChIP‐seq were H3K4me1 (Abcam ab8895), H3K4me3 (Abcam ab8580), H3K9ac (Active Motif 39137), H3K9me3 (Abcam ab8898), H3K27ac (Abcam ab4729), H3K27me3 (Abcam ab6002), H3K36me3 (Abcam ab9050), H3 (Abcam ab1791), CTCF (Active Motif 61311), and EBF1 (Sigma SAB2501166) and are listed in Appendix Table S4.