GSM8230194: E7T-seq_wt_5wo_rep2; Mus musculus; OTHER
Testis cells from mice at indicated age were obtained as described previously (ref. 1). Cells were embedded in plugs of low-melting point agarose to protect DNA from shearing, and DNA was liberated by treatment with SDS and proteinase K as described previously (ref. 1). In-plug overhang removal with S1 endonuclease for S1-seq, exonuclease VII and exonuclease T for Exo7/T-seq, or exonuclease T for ExoT-seq, and adaptor ligation were performed as described previously (ref. 1, 2, 3) with minor modifications. In brief for S1-seq, plugs were washed in 0.5 ml of modified S1 buffer (50 mM sodium acetate, 280 mM NaCl, 4.5 mM ZnSO4, pH 4.7) for 30 min four times, treated with 9 units of S1 nulcease in 500 µl of modified S1 buffer at 37°C for 20 min and washed once with 1ml TE. For Exo7/T-seq, plugs were washed in 1 ml of exonuclease VII buffer for 15 min twice, treated with 50 units of exonuclease VII in 100 µl of exonuclease VII buffer at 37°C for 60 min and washed once with 1ml TE and followed by exonulcease T treamtment. For ExoT-seq, plugs were washed in 1 ml of NEBuffer 4 for 15 min 3 times, treated with 75 units of exonuclease T in 100 µl of NEBuffer 4 at 24°C for 90 min and washed 3 times with 8 ml of TE for 15 min. The nuclease treated ssDNA ends were further polished with T4 polymerase before the first adaptor ligation as previously described (ref. 1, 2, 3). After ligation to biotinylated adaptors, DNA was purified from the agarose, sheared by sonication, purified with streptavidin, ligated to second-end adaptors, amplified, and sequenced as described previously (ref. 1, 2, 3). (ref. 1) Yamada et al., Molecular structures and mechanisms of DNA break processing in mouse meiosis. Genes Dev 34, 806-818 (2020). (ref. 2) Mimitou et al., S. A global view of meiotic double-strand break end resection. Science 355, 40-45 (2017). (ref. 3) Mimitou et al., S1-seq Assay for Mapping Processed DNA Ends. Methods Enzymol 601, 309-330 (2018). After Qubit quantification and quality control by Agilent BioAnalyzer, libraries were pooled equimolar and run on an Illumina sequencer. A spike-in of PhiX was added to the run to increase diversity when necessary and for quality control purposes. Base calls were perfomed using bcl2fastq software v.2.20 or bcl-convert software v.3.9.3 to 3.10.5 for samples sequenced on Illumina NextSeq 500, NextSeq 2000, or NovaSeq 6000.  Reads were trimmed and filtered by Trim Galore version 0.6.6 with the arguments --paired --length 15 <http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/>. Sequence reads were mapped onto the mouse reference genome (mm10) by bowtie2 version 2.3.5.1 (ref. 1) with the arguments -N 1 -X 1000. (ref. 1) Langmead et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009). Duplicated reads were removed by Picard <https://broadinstitute.github.io/picard/>. Uniquely and properly mapped reads (MAPQ ≥ 20) were extracted by samtools with the arguments -q 20 <http://www.htslib.org/>. Reads for S1, Exo7/T, or ExoT-seq were counted at which a nucleotide next to biotinylated adaptor DNA was mapped (this corresponds to a position of ssDNA). Maps were analyzed using R version 4.0.3 <http://www.r-project.org>. S1-seq, Exo7/T-seq, ExoT-seq