Smarter thruplex dna seq kit

Total RNA from sorted target cell populations was isolated using
TRIzol LS (Invitrogen) followed by DNase I (QIAGEN) treatment and cleanup
with RNeasy Plus Micro kit (QIAGEN; 74034). RNA quality was assessed using
Pico Bioanalyser chips (Agilent). RNASeq libraries were prepared using the
Clontech Smart-Seq Ultra low RNA kit (Takara Bio) for cDNA preparation,
starting with 550 pg of total RNA, and 13 cycles of PCR for cDNA
amplification. The SMARTer Thruplex DNA-Seq kit (Takara Bio) was used for
library prep, with 7 cycles of PCR amplification, following the
manufacturer’s protocol. The amplified library was purified using
AMPure beads (Beckman Coulter), quantified by Qubit and QPCR, and visualized
in an Agilent Bioanalyzer. The libraries were pooled equimolarly, and run on
a HiSeq 4000, as paired end reads, 50 nucleotides in length.

Active status of chromatin was determined by histone 3 lysine 27 acetylation (H3K27Ac) levels using ChIP-seq. H3K27Ac ChIP assay was conducted with 5 μg of anti-H3K27Ac antibody (Active Motif, 39133) using the protocol described above. Sequencing libraries were prepared with 3 ng each of H3K27Ac ChIP DNA and input sample using SMARTer ThruPLEX DNA-seq kit (Takara, R400675). Libraries were sequenced with single-end (SE) 75 cycles on an Illumina Nextseq 500 system at the Broad Institute of Harvard and MIT and the reads were aligned to human reference genome hg19 using Burrows-Wheeler Alignment (BWA) tool^{44 (link)}. Genome-wide coverage was calculated after extending to 200 bases (approximate fragment size) and averaged over 25 bp windows using igvtools^{45 (link)}. Coverage was then normalized and scaled using RSeqC (http://rseqc.sourceforge.net/#normalize-bigwig-py). ChIP-seq peaks were called using MACS2 2.0.10.20120913.

ChIP-seq was performed on three to four independent biological replicates. Chromatin was prepared from both cell lines/tumors and immunoprecipitated as described for qPLEX-RIME above. After washing of the beads, chromatin was eluted and decrosslinked by incubating over night at 65 °C in elution buffer (50 mM TrisHCl pH8, 10 mM EDTA, 1% SDS). Samples were treated with RNase A (20 ng/ml) for 30 min-1 h followed by proteinase K (200 ng/ml) for 1-2 h before DNA was purified by phenol-chloroform extraction. Purified DNA was subjected to library preparation using the SMARTer ThruPLEX® DNA-Seq Kit (TaKaRa, R400676) and DNA HT Dual Index Kit – 96N Set A (TaKaRa, R400660) followed by Illumina next generation sequencing to reach approximately 30 M reads per sample.

Cells treated with either DMSO or VTP50469 were crosslinked in 1% methanol-free formaldehyde (ThermoFisher) for 7-10 min at room temperature, followed by quenching of remaining formaldehyde with 100 mM Tris pH8.0 and 25 mM Glycine. Cytoplasm was stripped using 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM EDTA, 1% SDS for 10 min at ambient temperature followed by precipitation of nuclei by centrifugation at 10,000 ×g. The nuclei were then resuspended in 66 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM EDTA, 1.7% Triton X-100, 0.5% SDS and sheared using E100S (Covaris) to chromatin fragments of 200-400 base-pair DNA size. 1-5 μg of chromatin was used per immunoprecipitation with anti-Menin (Bethyl), MLLn (Bethyl), DOT1L (Cell Signaling) antibodies and protein-A magnetic beads (Dynal). Immunoprecipitated DNA fragments were eluted and de-crosslinked in 100 mM NaHCO₃, 100 mM NaCl, 1% SDS, and quantified by TapeStation (Agilent) and Qubit (ThermoFisher). 1-10 ng of DNA was used in preparation of Illumina-compatible libraries using SMARTer ThruPLEX DNA-Seq Kit (Takara) followed by sequencing using NextSeq550 (Illumina) to obtain 1-5×10⁷ unique sequencing paired-end tags.

ChIP-seq was performed in MV4;11 and MOLM13: cells were crosslinked in 1% methanol-free formaldehyde (ThermoFisher) for 7 min at room temperature followed by quenching of the formaldehyde using 100 mM Tris pH 8.0 and 250 mM Glycine. Cells were then lysed using 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM EDTA, 1% SDS for 10 min at ambient temperature and chromatin was collected by centrifugation at 10,000 3xg for 10 min. Chromatin was then resuspended in 66mM Tris-HCl pH 8.0, 100mM NaCl, 5mM EDTA, 1.7% Triton X-100, 0.5% SDS and sheared using an E100S sonicator (Covaris) to chromatin fragments of 200–400 base-pair DNA size. Sheared chromatin from the 20 million cells was used in each immunoprecipitation using the respective antibodies: anti-MENIN (Bethyl Cat#A300–105A), KMT2A/MLL1 (Bethyl Cat#A300–086A), MEIS1 (Abcam Cat#ab19867) and IKAROS (Cell Signaling Cat#14859S) antibodies and protein-A magnetic beads (Dynal). Immunoprecipitated DNA fragments were eluted and de-crosslinked in 100 mM NaHCO₃, 100 mM NaCl, 1% SDS, and quantified by TapeStation 4200 (Agilent) and Qubit (ThermoFisher). 1–10 ng of DNA was used in preparation of Illumina compatible libraries using SMARTer ThruPLEX DNA-Seq Kit (Takara) followed by sequencing using NextSeq550 (Illumina) to obtain paired-end reads (R1: 37bp, R2: 37bp).