Project description:gDNA was estracted and digested with cocktail of restrict enzymes MseI, DdeI, AluI, MboI, incubated at 37°C for 6 h. After purified by phenol/chloroform extraction, fragmented DNA was resuspended by TE, and quantified by Qubit 3.0 (Invitrogen). For each sample, 20 μg input DNA was immunoprecipitated overnight at 4°C in a shaker, with 1× DRIP binding buffer (10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) and 10 μg S9.6 antibody (ATCC, HB-8730). After adding 50 μl Dynabeads Protein G (Invitrogen, 10004D) and incubated for 4 h, the beads with antibody were washed by 1× DRIP binding buffer for 4 times at room temperature, each wash takes 10 min. Added 250 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) and 5 U Proteinase K to beads/antibody complexes, incubated for 40 min in an Eppendorf ThermoMixer at 55°C, 1,000 rpm. Purified by phenol/chloroform extraction, and moved supernatant to a new tube, followed by adding 1/10 volume 3 M NaAc, 1 μl GlycoBlue (Invitrogen) and 1 volume isopropanol, precipitated at -20°C for 3 h and then centrifuged. After washed by 70% ethanol and dried, the DRIPed DNA pellet was resuspended by 75 μl low EDTA TE buffer (10 mM Tris-HCl 8.0, 0.1 mM EDTA). NGS libraries were constructed using Accel-NGS® 1S Plus DNA Library Kit (Swift Biosciences).
Project description:Cells were washed with cold PBS twice and scraped from the plates, then centrifuged at 1000 rpm for 5 min to pellet cells. Washed cells were lysed with 400 L of cell lysis buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 0.15% NP-40 and proteinase inhibitor cocktail) and incubated on ice for 10 min. The suspension was carefully add at the top of sucrose buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 24% sucrose), and centrifuged at 3200g for 10 min to collect nuclear pellets. Pellets were resuspended in 250 L of Glycerol buffer (20 mM Tris-HCl pH7.5, 75 mM NaCl, 0.5 mM EDTA pH8.0, 50% glycerol), then then immediately add 250 μl Nuclear lysis buffer (10 mM Tris-HCl pH7.5, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA pH8.0, 1% NP-40, 1M urea). Mix by vortexing for 4 s and incubate on ice for 2 min. Centrifuge the lysate at 13,000 × g for 2 min to precipitate the chromatin–RNA complex. Briefly rinse the chromatin pellets with PBS-EDTA. RNA was extracted with Trizol reagent. RNA libraries were prepared according to manual of SMARTer smRNA-Seq Kit for Illumina (Clontech, 635031).
Project description:The chromatin fraction was isolated from 5x107 HeLa cells. Chromatin was digested in 100 mL of MNase (40 units/ mL ) reaction buffer for 3 min at 37 °C in a thermomixer (1,400 rpm). After addition of 10 L EGTA (25mM) to inactivate MNase, soluble digested chromatin was collected by 13,000 rpm centrifuge for 5 min. 10 mg of Pol II antibody was conjugated to magnetic protein A beads. The supernatant was diluted with 400 mL of NET-2 buffer and pSer2-Pol II antibody-conjugated beads were added. Incubated the IP reaction at 4 °C for 1 hr. The beads were washed with 1 mL of NET-2 buffer six times and 100 mL of 1xPNKT (1xPNK buffer and 0.05% Triton X-100) buffer once. Washed beads were incubated in 50 mL PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK (NEB)) in Thermomixer at 37 °C, 1,400 rpm for 6 min. Beads were washed with 1 mL of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was suspended in urea Dye (7M Urea, 1xTBE, 0.1% BPB and 0.1% XC) and resolved on 6% TBU gel (Invitrogen) at 200 V for 5 min. Gels with 30-160 nt RNAs were collected. 0.2 mL tube was prepared with holes made with 25G needle and placed in a 1.5 mL tube. Gel fragments were placed in the layered tube and broken down by centrifugation at 12,000 rpm for 1 min. The small RNAs were eluted from gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) at 25 °C for 1 hr in Thermomixer (900 rpm). Eluted RNA was purified with SpinX column (Coster) with 2 glass filters (Millipore) and the flow-through RNA was ethanol precipitated.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to analysis the differiational genes and pathways in MYCWT+shCtrl, MYCWT+shTRIB3, MYCT58A+shCtrl and MYCT58A+shTRIB3 lymphoma cells by using NGS-derived lymphoma transcriptome profiling (RNA-seq). Methods: MYCWT+shCtrl, MYCWT+shTRIB3, MYCT58A+shCtrl and MYCT58A+shTRIB3 cells' mRNA profiles were generated by deep sequencing, in triplicate, using Illumina HiSeq 4000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with following methods: Alignment by using HISAT2 v2.1, IGV was used to to view the mapping result by the Heatmap, histogram, scatter plot or other stytle, FPKM was then calculated to estimate the expression level of genes in each sample, DEGseq v1.18.0 was used for differential gene expression analysis between two samples with non biological replicates and Function Enrichment Analysis including GO enrichment analysis and KEGG . Conclusions: Our study represents the first detailed analysis of MYCWT+shCtrl, MYCWT+shTRIB3, MYCT58A+shCtrl and MYCT58A+shTRIB3 cells' transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
Project description:In this study, to obtain the biological impact of the mutated U2AF35, HeLa cells were retrovirally transduced with either mock, wild-type or S34F mutant of U2AF35, and Exon array was performed. Exon level analysis was performed for mock, wild-type U2AF35 or S34F mutant transduced HeLa in triplicate.
Project description:The newly discovered dynamic N6-methyladenosine (m6A) modification plays a critical role in gene expression from a post-transcriptional level. we profiled the transcriptome-wide m6A modification in mRNAs and lncRNAs in non-targeting control A375 cells(NC) and METTL3 knockdown A375 cells(shMETTL3). Methylated RNA immunoprecipitation sequencing results revealed that the RNA m6A modification is conserved. METTL3 knockdown altered the expression of RNAs m6A modified sites in A375 cells. Finally, we show that the transcriptome-wide m6A alterations occurring in mRNAs and lncRNAs following METTL3 knockdown suggest this process plays important regulatory roles during A375 growth. This study provides a framework for applying the m6A modification regulated by METTL3 to melanoma research.
Project description:To explore the target genes of long noncoding RNA lncTCF7, we established lncTCF7-silenced HCC primary CSC cells and conducted transcriptome microarray analysis. We used microarrays to identify distinct gene expression underlying shCtrl and shlncTCF7 of hepatocellular carcinoma sample stem cells. We cultured shlncTCF7 and shCtrl cells from hepatocellular carcinoma (HCC) clinical sample, then hybridized on Affymetrix microarrays. We sought to identify distinct target genes of lncTCF7 in liver cancer stem cells (CSCs).