Project description:In MINUTE-ChIP, native chromatin is fragmented using Micrococcal nuclease, and subsequently blunted and ligated to double-stranded DNA adaptors that include a T7 RNA polymerase promoter and a sample barcode sequence. Finally, samples are combined and subsequent ChIP reactions are performed with the pooled samples. ChIP material is prepared into an Illumina-compatible library using linear amplification by virtue of T7 RNA polymerase, reverse transcription and a low-cycle library PCR amplification. Native MINUTE-ChIP is based on Mint-ChIP, developed by the Bernstein lab (van Galen et al., 2016; PMID: 26687680). We have introduce unique molecule (UMI) counting and paired-end mapping of the chromatin fragments to this method, which we then termed MINUTE-ChIP for multiplexed indexed unique molecule T7 amplification end-to-end sequencing. Here, we generate a standard curve for H3K27me3 and demonstrate that MINUTE-ChIP has a large linear dynamic range, thus MINUTE-ChIP quantitation is proportional to real quantities.
Project description:In crosslinked MINUTE-ChIP, formaldehyde-fixed chromatin is fragmented using sonication, blunted and ligated to double-stranded DNA adaptors that include a T7 RNA polymerase promoter and a sample barcode sequence. Finally, samples are combined and subsequent ChIP reactions are performed with the pooled samples. ChIP material is prepared into an Illumina-compatible library using linear amplification by virtue of T7 RNA polymerase, reverse transcription and a low-cycle library PCR amplification. Here, we demonstrate a MINUTE-ChIP for Nanog from formaldehyde-fixed cells using fragmentation by sonication.
Project description:Ribosome profiling (Ribo-Seq) and RNA-Seq analysis of Saccharomyces cerevisiae with and without 10 minute glucose starvation to determine effects on translation.
Project description:Heat stress (HS) could damage animal reproduction, while the mechanisms are still required to be better understood. Here, we showed that incubation of porcine immature Sertoli cells (iSCs) at 43 °C for 30 minutes followed 36h recovery under normal culture could inhibit the proliferation, promote apoptosis and increase the lactate production. Then, we profiled mRNA expression by RNA sequencing on Control (before HS), HS0.5 (0h after HS) and HS0.5-R36 (36h recovery after HS) groups of iSCs. We identified 126 (92 up- and 64 down-regulated), 3372 (2076 up- and 1296 down-regulated) and 3096 (1772 up- and 1324 down-regulated) differentially expressed (DE) mRNAs respectively in HS0 vs. Control, HS0-R36 vs. HS0 and HS0-R36 vs. Control comparisons. Gene ontology (GO ) enrichment found multiple significantly enriched biological pathways involved in different comparisons, including cellular response to heat, heat shock protein binding, regulation of cellular response to stress, RNA polyadenylation, sterol biosynthetic process, positive regulation of stress-activated MAPK cascade, chaperone-mediated protein folding, ATPase activity, cell cycle process and DNA replication etc. KEGG analysis showed the changed pathways, including cell cycle, MAPK, P53, PI3K-AKT, GnRH, HIF1, Wnt, cAMP signal pathways and Glycolysis/Gluconeogenesis etc. RT-qPCR validation of 9 DEGs (DNAJB1, TRAF6, INSIG1, GADD45G, HDAC6, FKBP4, SERPINE1, PFKP and GALM) showed the expression change trend of most DEGs (except TRAF6) consistent with the RNA-seq. Moreover, 6 of HSPs (HSPA6, HSPB1, HSPD1, HSP90AA1, HSP90AB1 and HSPH1) were validated by RT-qPCR and 5 of 6 (except HSP90AB1) showed the similar expression trend to RNA-seq results. Further validation of HSP90AA1 on protein level via immunostaining and westernbloting showed similar expression trend to RT-qPCR and RNA-seq. Taken together, HS could extensively change the global transcriptome, especially HSPs, to affect proliferation, apoptosis and metabolite production of pig iSCs.