DOI: 10.57760/sciencedb.o00019.00001
title: Identification and characterization of BEND2 as a novel and key regulator of meiosis during mouse spermatogenesis
title: Identification and characterization of BEND2 as a novel and key regulator of meiosis during mouse spermatogenesis

description: Procedures for BEND2 ChIP-seqApproximately 60 mg of testicular tissues from mice at 15 or 40 dpp was disaggregated by Dounce homogenization and the chromatin was crosslinked in PBS containing 1% formaldehyde for 10 min at RT. Fixation of chromatin was halted with a 1.25 M glycine solution and washed three times with cold PBS. Cells was lysed for 30 min on ice by adding cell-lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS, and fresh 1 mM PMSF and protease inhibitor were added before use). Chromatin was then sonicated for 20 s at 25% power in 30-s pulses for 20 cycles, cellular debris was removed by centrifugation, and the supernatant was precleared by IgG for 2 h at 4°C. About 1/20th of the chromatin was saved as an input sample, and the remainder was diluted to 10x volume with IP dilution buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.01% SDS), and incubated with 10 μg of antibody for 2 h at 4°C. Protein A/G (10001D, 10003D, Invitrogen) Dynabeads were added to capture targeted chromatin overnight. Beads were washed with a four-step wash buffer (low-salt wash buffer, 20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, and 0.1% SDS; high-salt wash buffer, 20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, and 0.01% SDS; LiCl wash buffer, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.25 M LiCl, 1% NP-40, and 1% deoxycholic acid; TET buffer, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA, and 0.1% Tween 20); beads were washed twice for each step. After the chromatin was eluted from the beads by elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, and 1% Tween 20), cross-linking was reversed with 5 M NaCl at 65°C for 16 h; and RNA and protein were digested by adding RNase A and protease K for 2 h at 37°C and 45°C, respectively. Extracted DNA was used to construct a library with the NEBNext Ultra II DNA library Prep Kit for Illumina (E7645, NEB), and qualified libraries were sequenced with an Illumina Novaseq 6000 to obtain paired-end 150-nt reads.Procedures for H3K4me3 and H3K27me3 ChIP-seqFor histone modification, we preferd CUT&RUN method.We collected zygotene spermatocytes via FACS as described above, and CUT&RUN was primarily performed according to Henikoff et al. . In brief, before experimentation, 10 μl of concanavalin A beads (BP531, Bangs Laboratories) were washed twice with binding buffer, resuspended with 10 μl of binding buffer, and maintained on ice. For each sample, ~20,000 cells were suspended in wash buffer, incubated with prepared beads, and mixed for 10 min at RT. After discarding liquids, beads were incubated with antibody buffer on a Thermomixer at 4°C overnight. Beads were washed once with Dig-Wash buffer, resuspended with the same buffer containing pAG-MNase at a final concentration of 700 ng/ml (we purified the pAG-MNase according to methods described previously (91), and this mixture was incubated for three hours at 4°C. Beads were then washed twice with the Dig-Wash buffer, resuspended in the same buffer containing 2 mM CaCl2, vortexed, and placed on ice as soon as possible. After 30 min, a 2x stop buffer was added to quench the digested reaction, and it was incubated at 37°C for 30 min. The suspensions were collected and the DNA was extracted. We prepared the library using a NEBNext Ultra II DNA library Prep Kit for Illumina (E7645, NEB), and sequenced the qualified libraries with an Illumina Novaseq 6000 system to obtain paired-end 150-nt reads.Procedures for ATAC-seqZygotene spermatocytes were collected by FACS as described in CUT&RUN, and cells were washed twice with cold PBS. To prepare nuclei, cells were lysed with cold lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, and 0.1 % NP-40), maintained on ice for 10 min, and centrifuged at 500 x g for 5 min at 4°C. After carefully removing the suspension, we resuspended the pellet in the transposase reaction mix (TD501, Vazyme) and incubated it at 37°C for 10 min. Fragments were then immediately purified with 2 x AMPure XP beads (A63881, Beckman), and library amplification was performed using the TruePrep DNA Library Prep Kit V2 for Illumina (TD501, Vazyme).Procedures for RNA-seqWe collected leptotene or zygotene spermatocytes via FACS (89). Briefly, the testes from one adult WT mouse or from three adult KO mice were digested by two-step methods. Seminiferous tubules were segregated with collagenase I and DNase I, and then 0.25% trypsin and DNase I were used to obtain a single-cell suspension. Testicular cells isolated from WT and KO mice were then sorted by FACS after Hoechst 33342 staining. Different types of spermatocytes were then collected through Hoechst Blue and Hoechst Red channels, and RNA was prepared following the TRIzol (Invitrogen) protocol. All RNA libraries were constructed at the same time using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7760) according to the manufacturer’s recommendations, and oligo (dT) beads (NEB) were used to isolate poly (A) mRNAs.Analysis procedureChIP-seq raw reads were trimmed to remove the adapter sequence when converting to a fastq file, and the trimmed ChIP-seq reads were mapped to the UCSC mm10 genome using Bowtie2 (v2.4.1) with default parameters. For BEND2, peak calling was performed using Pepr with default parameters and the corresponding inputs as background, and peaks that mapped to blacklist regions were removed.Annotation of genomic locations and repeat types were generated using HOMER (v4.11), and heatmaps were generated using the command-line version of deepTools (v3.5.0) . Distribution of BEND2-binding sites on the chromosomes were generated by ChIPseeker (v1.24.0) , and HOMER (v4.11) was used with default settings to identify enriched motifs in BEND2 peaks. RepeatMasker and TSS-location files were downloaded from the UCSC website, and we achieved a Genome Browser view of the NGS data by using the command-line version of pyGenomeTracks. Our RNA-seq analyses followed a standard procedure that included mapping sequence reads to the mouse genome mm10 by using Bowtie2 and identifying differentially expressed genes by using the DESeq2 R package. For ATAC-seq, Paired-end reads were aligned with Bowtie2 using default parameters, and only uniquely mapping reads were retained for further analysis; PCR duplicates and blacklist-region reads were removed. Peak calling was executed using MACS2 (v2.2.7.1). Different gene clusters and heatmaps were then generated using the command-line version of deepTools (v3.5.0), and correlation analysis between the three clusters of genes by ATAC-seq and differentially expressed genes was based upon a hypergeometric distribution.
description: Procedures for BEND2 ChIP-seqApproximately 60 mg of testicular tissues from mice at 15 or 40 dpp was disaggregated by Dounce homogenization and the chromatin was crosslinked in PBS containing 1% formaldehyde for 10 min at RT. Fixation of chromatin was halted with a 1.25 M glycine solution and washed three times with cold PBS. Cells was lysed for 30 min on ice by adding cell-lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS, and fresh 1 mM PMSF and protease inhibitor were added before use). Chromatin was then sonicated for 20 s at 25% power in 30-s pulses for 20 cycles, cellular debris was removed by centrifugation, and the supernatant was precleared by IgG for 2 h at 4°C. About 1/20th of the chromatin was saved as an input sample, and the remainder was diluted to 10x volume with IP dilution buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.01% SDS), and incubated with 10 μg of antibody for 2 h at 4°C. Protein A/G (10001D, 10003D, Invitrogen) Dynabeads were added to capture targeted chromatin overnight. Beads were washed with a four-step wash buffer (low-salt wash buffer, 20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, and 0.1% SDS; high-salt wash buffer, 20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, and 0.01% SDS; LiCl wash buffer, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.25 M LiCl, 1% NP-40, and 1% deoxycholic acid; TET buffer, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA, and 0.1% Tween 20); beads were washed twice for each step. After the chromatin was eluted from the beads by elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, and 1% Tween 20), cross-linking was reversed with 5 M NaCl at 65°C for 16 h; and RNA and protein were digested by adding RNase A and protease K for 2 h at 37°C and 45°C, respectively. Extracted DNA was used to construct a library with the NEBNext Ultra II DNA library Prep Kit for Illumina (E7645, NEB), and qualified libraries were sequenced with an Illumina Novaseq 6000 to obtain paired-end 150-nt reads.Procedures for H3K4me3 and H3K27me3 ChIP-seqFor histone modification, we preferd CUT&RUN method.We collected zygotene spermatocytes via FACS as described above, and CUT&RUN was primarily performed according to Henikoff et al. . In brief, before experimentation, 10 μl of concanavalin A beads (BP531, Bangs Laboratories) were washed twice with binding buffer, resuspended with 10 μl of binding buffer, and maintained on ice. For each sample, ~20,000 cells were suspended in wash buffer, incubated with prepared beads, and mixed for 10 min at RT. After discarding liquids, beads were incubated with antibody buffer on a Thermomixer at 4°C overnight. Beads were washed once with Dig-Wash buffer, resuspended with the same buffer containing pAG-MNase at a final concentration of 700 ng/ml (we purified the pAG-MNase according to methods described previously (91), and this mixture was incubated for three hours at 4°C. Beads were then washed twice with the Dig-Wash buffer, resuspended in the same buffer containing 2 mM CaCl2, vortexed, and placed on ice as soon as possible. After 30 min, a 2x stop buffer was added to quench the digested reaction, and it was incubated at 37°C for 30 min. The suspensions were collected and the DNA was extracted. We prepared the library using a NEBNext Ultra II DNA library Prep Kit for Illumina (E7645, NEB), and sequenced the qualified libraries with an Illumina Novaseq 6000 system to obtain paired-end 150-nt reads.Procedures for ATAC-seqZygotene spermatocytes were collected by FACS as described in CUT&RUN, and cells were washed twice with cold PBS. To prepare nuclei, cells were lysed with cold lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, and 0.1 % NP-40), maintained on ice for 10 min, and centrifuged at 500 x g for 5 min at 4°C. After carefully removing the suspension, we resuspended the pellet in the transposase reaction mix (TD501, Vazyme) and incubated it at 37°C for 10 min. Fragments were then immediately purified with 2 x AMPure XP beads (A63881, Beckman), and library amplification was performed using the TruePrep DNA Library Prep Kit V2 for Illumina (TD501, Vazyme).Procedures for RNA-seqWe collected leptotene or zygotene spermatocytes via FACS (89). Briefly, the testes from one adult WT mouse or from three adult KO mice were digested by two-step methods. Seminiferous tubules were segregated with collagenase I and DNase I, and then 0.25% trypsin and DNase I were used to obtain a single-cell suspension. Testicular cells isolated from WT and KO mice were then sorted by FACS after Hoechst 33342 staining. Different types of spermatocytes were then collected through Hoechst Blue and Hoechst Red channels, and RNA was prepared following the TRIzol (Invitrogen) protocol. All RNA libraries were constructed at the same time using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7760) according to the manufacturer’s recommendations, and oligo (dT) beads (NEB) were used to isolate poly (A) mRNAs.Analysis procedureChIP-seq raw reads were trimmed to remove the adapter sequence when converting to a fastq file, and the trimmed ChIP-seq reads were mapped to the UCSC mm10 genome using Bowtie2 (v2.4.1) with default parameters. For BEND2, peak calling was performed using Pepr with default parameters and the corresponding inputs as background, and peaks that mapped to blacklist regions were removed.Annotation of genomic locations and repeat types were generated using HOMER (v4.11), and heatmaps were generated using the command-line version of deepTools (v3.5.0) . Distribution of BEND2-binding sites on the chromosomes were generated by ChIPseeker (v1.24.0) , and HOMER (v4.11) was used with default settings to identify enriched motifs in BEND2 peaks. RepeatMasker and TSS-location files were downloaded from the UCSC website, and we achieved a Genome Browser view of the NGS data by using the command-line version of pyGenomeTracks. Our RNA-seq analyses followed a standard procedure that included mapping sequence reads to the mouse genome mm10 by using Bowtie2 and identifying differentially expressed genes by using the DESeq2 R package. For ATAC-seq, Paired-end reads were aligned with Bowtie2 using default parameters, and only uniquely mapping reads were retained for further analysis; PCR duplicates and blacklist-region reads were removed. Peak calling was executed using MACS2 (v2.2.7.1). Different gene clusters and heatmaps were then generated using the command-line version of deepTools (v3.5.0), and correlation analysis between the three clusters of genes by ATAC-seq and differentially expressed genes was based upon a hypergeometric distribution.