Ontology highlight
ABSTRACT:
PROVIDER: PRJNA756718 | ENA |
REPOSITORIES: ENA
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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.
2019-10-15 | GSE134102 | GEO
Project description:Different lesion types were microdissected out from snap-frozen white matter tissue. Brain nuclei were fluorescence-activated nuclear sorted. Gel beads-in-emulsion was generated, barcoded, reverse transcribed followed by library construction and sequencing.
2024-06-21 | GSE231585 | GEO
Project description:The impact of chirality on immune response has attracted great interest in cancer vaccine research recently. However, the study of chiral synthetic polypeptide hydrogels as cancer vaccines as well as of the impact of biomaterials itself for antitumor immunotherapy has rarely been reported. Herein, we demonstrated the key role of residue chirality of polypeptides hydrogels in antitumor immunity and local immune microenvironment regulation. Compared to poly(γ-ethyl-L-glutamate)-based hydrogels (L-Gel), poly(γ-ethyl-D-glutamate)-based hydrogels (D-Gel) induced greater immune cell infiltration. Unexpectedly, D-Gel caused higher levels of suppressive markers on antigen-presenting cells and even induced stronger T cells exhaustion than L-Gel. Finally, D-Gel established a local chronic inflamed and immunosuppressive microenvironment and showed insufficient anti-tumor effects. Conversely, the milder host immune responses induced by L-Gel led to more effective tumor inhibition. This study provides new insights on the role of residue chirality in the regulation of local immune microenvironment and affecting antitumor immune response.
2024-03-14 | GSE261299 | GEO