title: GSM8974455: LG_JUNB-KD_RNAseq_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: ChIP-seq: Chromatin (8 μg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were precipitated using 4 μg of antibody against H3K27Ac (Active Motif, Cat# 39133) or 10 μg of antibody again JUNB (Activemotif, Cat#39549). The complexes were washed, eluted from the beads with 1% SDS buffer and subjected to RNase and proteinase K treatment overnight at 65°C. The ChIPed DNA was purified by phenol-chloroform extraction and ethanol precipitation. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K in a similar way as ChIPed DNA. ATAC-seq: Cells grown in tissue culture were pretreated with 200 U/ml DNase (Worthington) for 30 min at 37 °C to remove free-floating DNA and to digest DNA from dead cells. This medium was then washed out, and the cells were resuspended in cold PBS. After the cells were counted, 50,000 cells were resuspended in 1 ml of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water). Cells were centrifuged at 500 g for 5 min at 4 °C. Supernatant was carefully aspirated. Cell pellets were then resuspended in 50 μl of ATAC-seq RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin by pipetting up and down three times. This cell lysis reaction was incubated on ice for 3 min. After lysis, 1 ml of ATAC-seq RSB containing 0.1% Tween-20 was added, and the tubes were inverted to mix. Nuclei were then centrifuged for 10 min at 500 g at 4 °C. Supernatant was removed and nuclei were resuspended in 50 μl of transposition mix (25 μl 2× TD buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2), 2.5 μl transposase (100 nM final), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water) by pipetting up and down at least five times. Transposition reactions were incubated at 37 °C for 30 min in a thermomixer with shaking at 1,000 r.p.m. Reactions were cleaned up with Zymo DNA Clean and Concentrator-5 kit. RNA-seq: Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. H3K27ac HiChIP: Biotin-labeled proximally-ligated chromatin was prepared using the Arima-HiC kit (Arima Genomics Cat # A510008). Briefly, the crosslinked chromatin was initially digested using a restriction enzyme (RE) cocktail. The 5'-overhangs were subsequently filled in and labeled with a biotinylated nucleotide. Subsequently, the spatially proximal digested ends of DNA were ligated. The resulting proximally-ligated chromatin was fragmented using the Diagenode Bioruptor Pico instrument, bound to an H3K27ac antibody (Abcam Cat # ab4729) overnight and immunoprecipitated on Protein A magnetic beads (Thermo Cat # 10002D). The immunoprecipitated chromatin was reverse crosslinked and purified using the Arima-HiC kit. Subsequently, the Illumina-compatible sequencing libraries were prepared by initial enrichment for biotin using C1 streptavidin beads (Thermo Cat # 65001). The samples were tagmented on-bead utilizing Tagment DNA Enzyme and Buffer (Illumina Cat # 20034197). Subsequently, DNA was ready for PCR enrichment. ChIP-Seq libraries were generated from the ChIP-DNA using a custom Illumina library type on an automated. system (Apollo 342, Wafergen Biosystems/Takara). ChIP-Seq libraries were sequenced on Illumina NextSeq 500 as 75-nt single end reads. Adapter sequences were not trimmed during demultiplexing. ATAC-seq: Libraries were prepared as the description (Buenrostro et al., 2015). RNA-seq: After extracting the total RNA from samples, mRNA and non-coding RNAs are enriched by removing rRNA form the total RNA with kit. By using the fragmentation buffer, the mRNA and non-coding RNAs are fragmented into short fragments (about 200~700 bp), then the first-strand cDNA is synthesized by random hexamer-primer using the fragments as templates. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand cDNA. The double strand cDNA is purified with QiaQuick PCR extraction kit and then used for end-polishing. Sequencing adapters are ligated to the fragments , then the second strand is degraded using UNG(Uracil-N-Glyosylase)finally. The fragments are purified by Agarose gel electrophoresis and enriched by PCR amplification.The library products are ready for sequencing analysis via Illunima HiSeq 4000 or Hiseq X Ten. HiChIP: Tagmented DNA was PCR amplified utilizing NPM and the indexing primers from the Nextera XT DNA Library Prep kit (Illumina Cat # FC-131-1024). The PCR products were purified and size selected by 0.5X SPRI beads followed by 0.7X SPRI beads before sequencing.