Project description:Standard Micro-C protocols typically require millions of cells, which limits their application to rare cell populations. Here, we present an optimized low-input Micro-C workflow that requires only 100,000 cells. By comparing 30 G of sequencing data from 100,000 cells with that from 5 million G1E-ER4 cells, we demonstrate that all key architectural features—compartments, TADs, and chromatin loops—are reliably detected. Applying this method to investigate acute CTCF degradation, we observed the loss of loops and TAD boundaries in CTCF-degraded samples, consistent with previous reports. Our optimized protocol enables nucleosome-resolution 3D genome mapping for sample-limited studies.
Project description:Various test protocols to improve the 3' pull down transcript profiling protocol, aiming to produce a pipeline library prep protocol. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/ Protocol: Total RNA was extracted from zebrafish embryos using Trizol and DNase treated. Chemically fragmented RNA was enriched for the 3 ends by pulled down using an anchored polyToligo attached to magnetic beads. An RNA oligo comprising part of the Illumina adapter 2 was ligated to the 5 end of the captured RNA and the RNA was eluted from the beads. Reverse transcription was primed with an anchored polyT oligo with part of Illumina adapter 1 at the 5 end followed by 12 random bases, then an 8 base indexing tags, then CG and 14 T bases. An Illumina library with full adapter sequence was produced by 20 cycles of PCR. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:Set up and modification of Agilent standard protocols for a new custom 60-mer array for C. acetobutylicum. Keywords: Test of platform and protocols
Project description:RNA samples from human tissues (brain and liver) and cell lines (K562 and HL60) were used to assess RNA fragmentation, RNA fractionation, cDNA synthesis, and single versus multiple tag counting. Technical replicates and different cDNA synthesis protocols were compared. Though protocols employing polyA RNA selection generate the highest number of non-ribosomal reads and the most precise measurements for coding transcripts, such protocols were found to detect only a fraction of the non-ribosomal RNA in human cells. PolyA RNA excludes thousands of annotated and even more unannotated transcripts, resulting in an incomplete view of the transcriptome. Ribosomal-depleted RNA provides a more cost-effective method for generating complete transcriptome coverage. Expression measurements using single tag counting provided advantages for assessing gene expression and for detecting short RNAs relative to multi-read protocols. Detection of short RNAs was also hampered by RNA fragmentation.