title: Initiation for 20 C replicate 2
design: About 10 mg of tissue was homogenized using a pellet pestle motor (KONTES, #749540-0000) for 1 min in the lysis buffer (50 L / mg tissue). The lysis buffer was prepared by supplementing 0.1% NP-40, 0.1% Tween-20 and 0.01% digitonin to the resuspension buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl and 3 mM MgCl2). The lysates were let to stay on ice for 5 min, 10 L nuclear suspension was transfer into a micro centrifuge tube containing 200 L wash buffer (resuspension buffer supplemented with 0.1% Tween-20) precooled on ice, the tube was turned upside down for 10 times to thoroughly mix the content. The samples were centrifuged at 4 C, 500 g for 10 min. The supernatant was carefully removed using a pipette and the nuclei were subjected to library construction using TruePrep DNA Library Prep Kit V2 for Illumina based on Tn5 transposase (Vazyme, #TD501-02) following the manufacturers instructions. The sequencing libraries were multiplexed using TruePrep Index Kit V3 for Illumina (Vazyme, #TD203). After PCR amplification, 200-700 bp fragments were selected by two rounds of size selection (0.55 / 1) using VAHTS DNA Clean Beads (Vazyme, #N411). The libraries were sequenced paired-end for 150 bp using a NovaSeq 6000 System (Illumina) at the Analysis and Testing Center at Institute of Hydrobiology (IHB), Chinese Academy of Sciences.