GSM6450067: SC-β_Input; Homo sapiens; ChIP-Seq
For ChIP-seq, H9 cells and their in vitro differentiated organoids were collected, fixed with 1% formaldehyde and homogenized to prepare for the ChIP-Seq libraries. After cell lysis, whole nuclear lysates were sonicated to shear the chromatin into fragments with an average size of around 200-500 bp in length. Chromatin was precleared with Protein A or G Dynabeads at 4°C for 2 hours and precipitated with antibody overnight at 4°C. Immunoprecipitated chromatin was tagmented with Tn5 transposes mixture on beads and then eluted, reverse crosslinked and purified by standard methods, followed by PCR amplification. For ATAC-seq, H9 cells and their in vitro differentiated organoids were collected for performing the OMNI-ATAC protocol. For OMNI-ATAC, organoids were homogenized with electric homogenizer for 10 seconds into small clusters of cells to perform standard OMNI-ATAC protocol. Briefly, cells were washed with PBS and nuclear membrane was disrupted by soaking in lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin) on ice, nuclei was washed once in cold lysis buffer without NP40 or digitonin, and then incubated in Tn5 transposes mixture (25 ul 2x TD buffer, 2.5 ul transposase (100nM final), 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O) at 37°C for 20 min with occasional shaking. After the reaction is over, DNA was directly extracted by Minelute Kit (Qiagen). Purified tagmented DNA were used to make Illumina Nextera libraries. For RNA-Seq, total RNA was isolated from H9 cells and their in vitro differentiated organoids using Trizol reagent (Invitrogen) and ribosomal RNA was removed using the Ribo Minus Transcriptome isolation kit (Invitrogen, K1550). Strand-specific RNA library prepared by TruSeq Stranded mRNA Library Prep Kit was amplified with barcoded primers for Illumina sequencing. For CUT&Tag, H9 cells and their in vitro differentiated organoids were harvested, gently homogenized with pestle A for standard library preparation. Briefly, cells were washed twice in 1.5 mL Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 × Protease inhibitor cocktail, EDTA free), immobilized to concanavalin A coated magnetic beads (Bangs Laboratories), and then resuspended in 50 μL Dig-wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 × Protease inhibitor cocktail, 0.05% Digitonin) containing 2 mM EDTA. After sequential incubation in the primary antibody of CHD4 (abcam, #ab72418, diluted 1:50 in 50 μL of Dig-Wash buffer) , the appropriate secondary antibody (diluted 1:100 in 100 μL of Dig-Wash buffer) and a 1:200 dilution of pAG-Tn5 (gift from Henikoff Lab) in Dig-300 Buffer (0.05% digitonin, 20 mM HEPES, pH7.5, 300 mM NaCl, 0.5 mM Spermidine, 1 × Protease inhibitor cocktail) , bead-bound cells were resuspended in 50μL tagmentation buffer (10 mM MgCl2 in Dig-wash Buffer) to tagment genomic DNA. The tagmented DNA was cleaned up by 1.5 × Ampure XP beads (Beckman Counter), amplified with Illumina Nextera barcoded primers, purified by 1.1 × Ampure XP beads for Illumina sequencing. Truseq libraries were constructed using the standard protocol. Genomic fragments were end repaired (NEBNext End Repair Module), A-tailed by adding adenosine to the 3' ends of fragment using Klenow fragment (3' to 5' exo minus, New England Biolabs), and adaptors were ligated at room temperature for 1 hr with T4 DNA ligase (New England Biolabs). Libraries were amplified with Illumina primers using the KAPA SYBR FAST qPCR Master Mix. Nextera libraries were amplified with Nextera barcoded primers using the KAPA SYBR FAST qPCR Master Mix. Illumina libraries were sequenced paired-end at 50 bp length on Illumina Hiseq2500  v4 or NovaSeq  6000  instruments.