title: GSM9019997: Clec3b-tdT⁺ cells (Input); Mus musculus; ChIP-Seq
library_name: GSM9019997
library_construction_protocol: Clec3b-tdT⁺ cells were cross-linked with 1% formaldehyde for 10 min at room temperature, and the reaction was quenched by the addition of glycine. Cells were washed twice with ice-cold PBS and harvested by scraping. Chromatin was fragmented by sonication in SDS lysis buffer using a Bioruptor (Scientz-IID, Scientz) to generate DNA fragments of approximately 100–500 bp. After preclearing with protein G agarose beads, the lysates were incubated overnight at 4 °C with either 2 µg of control IgG (CST, 2729S) or 2 µg of anti-KLF4 antibody (abcam, ab214666) for immunoprecipitation. Immune complexes were captured with protein G agarose beads, and cross-links were reversed by incubation at 65°C overnight. The sequencing libraries of immunoprecipitated chromatin DNA were generated with varied index label by NEBNext® UltraTM DNA Library Prep Kit for Illumina (NEB, USA, cat. E7645L). Add End Repair Reaction Buffer (10X) and End Prep Enzyme Mix for end repair by the following condition: 20°C for 30min, 65°C for 30min. And then NEBNext Adaptor and Blunt/TA Ligase Master Mix was added to ligate adaptor at 20°C for 15min. USER Enzyme was added to remove base U of adaptor at 37°C for 5min. After purification with beads, products are amplified with PCR by the following conditions: initial denaturation at 98℃ for 10 sec; denaturation at 98℃ for 10 sec, annealing at 65℃ for 30 sec, and extension at 72℃ for 30 sec. The library size was selected and then purified with AMPure XP Beads. Qubit was used for library quantification and High-sensitivity DNA chip was used for test of library insert size.