title: Kupffer Cell mouse 1-rep1 (CRX1581001; CRA024256)
library_construction_protocol: The tissue blocks were put into ice-cold PBS and cut into 8 10 m sections with a vibratome. The sections were placed in a lysis buffer (10 mM Tris-HCl, pH 8.0 ,10 mM NaCl 0.2%, Tween-100, 1% Protease Inhibitor Cocktail, P8340, Sigma-Aldrich), incubated on ice for 20 minutes, and then washed with NEBuffer 2 (B7002S, NEB). The sections were reversed by SDS (SDS final concentration: 1.2%) in 65C for 1 hour and incubated with Triton X-100 (Triton X-100 final concentration: 1%) at 37C, for 15 min. The sample was digested with MboI restriction enzyme (R0147, 5 U/mL, NEB) in 37C overnight, end repaired with Klenow in 37C for 2 hours and ligated using T4 ligase in 25C for 8 hours. In the end, the sample was incubated with Exonuclease (100 Units/mL) for 5 min at 37C. The collected sample were incubated with proteinase K (1mg/mL) and SDS (1%) at 65C for 3h and followed by the addition of 5 M sodium chloride to 500 M and incubated at 65C overnight. The DNA was captured by 1.8 X Agencourt AMPure XP beads (A63881, Beckman). The DNA was resuspended in 10 mM Tris-HCl, and then Tn5 enzyme (TruePrep DNA Library Prep Kit V2 for Illumina, TD502/TD503, Vazyme) was added to cut the DNA and add the adaptor. The biotin-labeled chimeric molecules were captured by incubating with Dynabeads MyOne Streptavidin C1 beads (65001, Life Technologies) in room temperature for 2 hours. After incubating, beads were washed with 1X Tween wash buffer (0.05% Tween 20, 1 M NaCl, 0.5 mM EDTA-NaOH, 5 mM Tris-HCl, pH 8) on a Thermomixer at 55C for 5 min, repeated 4 times. After washing, the DNA on the beads was amplified using TruePrepTM DNA Library Prep Kit V2. And then, 0.55 X &0.75 X Agencourt AMPure XP beads were used to select the DNA with suitable size for next sequencing. The library was sequenced in NovaSeq 6000 platform.