Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference |
Identifiers | Additional information |
|
|---|---|---|---|---|---|
| Mouse strains | |||||
| C57BL/6JOlaHsd | Envigo | C57BL/6JOlaHsd | Named B6 | ||
| B10.MOLSGR(A)-(D17Mit58-D17Jcs11)/Bdm (RJ2) | Grey et al., 2009 | MGI:5319075 | Named RJ2 | ||
| B6;129P2 < Prdm9tm1Ymat>/J | Hayashi et al., 2005 | MGI:3624989 | Named Prdm9 KO |
||
| Spo11 < tm1Mjn> | Baudat et al., 2000 | MGI:2178805 | Named Spo11 KO |
||
| Hells < tm1a(EUCOMM)Wtsi/Ieg> | EUCOM Bradley et al., 2012 |
MGI:4431905 | |||
| C57BL/6 Tg(CAG-Flpo)1Afst | Kranz et al., 2010 | MGI:4453967 | |||
| C57BL/6 Tg(CMV-cre)1Cgn | Schwenk et al., 1995 | MGI:2176180 | |||
| Tg(Stra8-icre)1Reb/J <(Stra8-iCre)> | Sadate-Ngatchou et al., 2008 | MGI:3779079 | |||
| Tg(RP23-159N6*)23Bdm | Diagouraga et al., 2018 | MGI:5565212 | Named B6-Tg(YF) |
||
| Cell lines | |||||
| HeLa | ATCC | HeLa S3 ATCC CCL-2.2 | |||
| Yeast strains | |||||
| AH109 | James et al., 1996 | S. cerevisiae | |||
| Y187 | Harper et al., 1993 | S. cerevisiae | |||
| Recombinant DNA reagents | |||||
| PRDM9A-Flag-HA-Nt into retroviral pOZ-FH-N vector | This study | N/A | Vector from Addgene DB3781 | ||
| PRDM9A-Flag-HA-Ct into retroviral pOZ-FH-C vector | This study | N/A | Vector from Addgene cat# 32516 | ||
| pGAD GH for fusion to Gal4 activation domain, modified for Gateway cloning | Van Aelst et al., 1993 | Clontech No. 638853 | LEU2 marker | ||
| pAS2dd for fusion to Gal4 DNA-binding domain, modified for Gateway cloning | Fromont-Racine et al., 1997 | TRP1 marker | |||
| pB29 for PRDM9 (aa 1–511) expression fused to LexA for yeast two-hybrid screen | Hybrigenics | ||||
| Antibodies | |||||
| Guinea-pig anti-SYCP3 | Grey et al., 2009 | N/A | Home-made WB: 1/2000 IF: 1/500 |
||
| Rabbit anti-SYCP1 | Abcam | Cat# ab15090 RRID:AB_301636 | IF: 1/400 | ||
| Rabbit anti-DMC1 | Santa Cruz | Cat# scH100 RRID:AB_2277191 | IF: 1/200 | ||
| Goat anti-DMC1 | Santa Cruz | Cat# scC20 RRID:AB_2091206 | ChIP: 24 µg | ||
| Rabbit anti-HELLS | Novus | Cat# NB 100–278 RRID:AB_350198 | WB: 1/2000 IF: 1/200 |
||
| Mouse monoclonal anti-HELLS | Santa Cruz | Cat# sc46665 RRID:AB_627895 | IF: 1/100 | ||
| Mouse monoclonal anti-phospho-histone H2AFX (Ser139) | Millipore | Cat# MP05-636 RRID:AB_309864 | Named γH2AFX IF: 1/10000 |
||
| Rabbit anti-Gal4 activation domain (GAD) (Millipore, 06–283) | Now at Sigma-Aldrich Cat# ABE476 |
WB: 1/3000 | |||
| Rabbit anti-Gal4 DNA-Binding domain | Sigma–Aldrich | Cat# G3042 RRID:AB_439688 | WB: 1/2000 | ||
| Rat monoclonal anti-Tubulin [YOL1/34] | Abcam | Cat# ab 6161 RRID:AB_305329 | WB: 1/3000 | ||
| Rabbit anti-5hmC | Active Motif | Cat# AM 39791 RRID:AB_2630381 | hMeDIP: 5 µg | ||
| Rabbit anti-PRDM9 | Grey et al., 2017 | N/A | Home-made WB: 1/2000 IF: 1/200 ChIP: 4 µg |
||
| Rabbit anti-H3K4me3 | Abcam | ab8580 RRID:AB_306649 | ChIP: 4 µg | ||
| Goat anti-rabbit IgG-HRP | Pierce | Cat# 1858415 RRID:AB_1185567 |
WB: 1/10000 | ||
| Goat anti-Guinea-pig IgG-HRP | Jackson Immuno Research | Cat# 706-035-148 RRID:AB_2340447 |
WB: 1/3000 | ||
| Goat anti-rabbit IgG-Alexa 555 | Thermo Fisher Scientific | Cat# ab150078 RRID:AB_2535849 | IF: 1/400 | ||
| Goat anti-guinea-pig IgG-Alexa 488 | Thermo Fisher Scientific | Cat# ab150185 RRID:AB_2534117 | IF: 1/400 | ||
| Donkey anti-mouse IgG-Alexa 680 | Thermo Fisher Scientific | Cat# ab175774 RRID:AB_2534014 | IF: 1/100 | ||
| Donkey anti-mouse IgG-Alexa 647 | Thermo Fisher Scientific | Cat# ab150107 RRID:AB_162542 | IF: 1/400 | ||
| Oligonucleotides | |||||
| Genotyping Hells cKO mice, see Supplementary file 2 | This study | ||||
| RT-qPCR, see Supplementary file 3 | This study, Buard et al., 2009, Diagouraga et al., 2018 | N/A | |||
| Commercial assays or kits | |||||
| DeadEnd Fluorometric TUNEL System | Promega | Cat# G3250 | |||
| Anti-HA beads | Santa Cruz | Cat# sc-500773 | |||
| EZview anti-FLAG M2 Affinity Gel | Sigma–Aldrich | Cat# F2426 | |||
| hMeDIP Kit | Actif Motif | Cat# AM55010 | |||
| NEB Next Ultra Library Preparation Kit | New England Biolabs | Cat# NEB7370S | |||
| ChIP-IT High Sensitivity Kit | Actif Motif | Cat# AM53040 | |||
| MMLV-based retroviral transduction system | Nakatani and Ogryzko, 2003 | N/A | |||
| Chemical compounds | |||||
| HA peptide | Covance | Cat #PEP-101P-1000 | |||
| FLAG peptide | Sigma | Cat #F4799 | |||
| Optiprep Idoixanol |
Sigma–Aldrich | Cat# D1556 | |||
| Sytox Green | Thermo Fisher Scientific | Cat# S70020 | |||
| WIN 18466 | Tocris Bioscience | Cat# 4736 | Hogarth et al., 2013 | ||
| Retinoic Acid | Sigma–Aldrich | Cat# R2625 | |||
| Deposited data | |||||
| Mass spectrometry proteomics |
ProteomeXchange Consortium | Dataset identifier PXD017337 | |||
| NGS SSDS ChIP (DMC1) and hMeDIP | GEO | GSE145768 | |||
| Softwares and Algorithms | |||||
| Bowtie 2 | http://bowtie-bio.sourceforge.net/bowtie2/index.shtml | ||||
| Modified BWA algorithm | Khil et al., 2012 | N/A | |||
| Tim Galore! | https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ | ||||
| Bismark | https://www.bioinformatics.babraham.ac.uk/projects/bismark/ | ||||
| Bedtools suite | https://bedtools.readthedocs.io/en/latest/content/bedtools-suite.html | ||||