title: GSM4678055: Carrier treated Healthy Donor 1 at 72h; Homo sapiens; RNA-Seq
library_construction_protocol: NEB Ultra II PolyA RNAseq Isolation of mRNA and RNAseq Library Prep: Memory CD4+ T cells, including from BACH2WT/L24P patient samples, were isolated and activated with or without VitD treatment as described.  6x105 unactivated cells, or cells activated for 48 hrs, were pelleted at 300g for 5 min. RNA was stabilized by lysing cells in 100mL of RNAqueous lysis buffer and snap-freezing on dry-ice followed by storage at -80C and isolation of total RNA by the RNAqueous®-Micro kit (AM1931, Thermo Fisher Scientific) as per manufacturers guidelines. 1mg total RNA for each sample was subjected to NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490). Resulting mRNA was prepared for RNA-seq using a NEB Ultra II RNA Sequencing Library Prep Kit (New England Biolabs) as per the manufacturer's guidelines. H3K27Ac, c-JUN, and IgG CUT&RUN: In situ CUT&RUN sequencing was performed on memory CD4+ T cells activated with anti-CD3+anti-CD28 in the presence of carrier or VitD for 0.75 hr, 48 hrs and 72 hrs using the published protocol of Skene et al. 2018 (Nature Protocols, DOI: 10.1038/nprot.2018.015). Rabbit antibodies targeting H3K27Ac (ab4729, Abcam), c-JUN (60AB, Cell Signaling Technologies), and non-specific IgG (31235, Thermo Fisher Scientific) were used, together with pAG-MNase (123461, Addgene) for small DNA fragment generation. All chemicals required for CUT&RUN buffers were purchased from Sigma-Aldrich and Thermo Fisher Scientific. Once in solution, isolated DNA fragments were then immediately prepared for Illumina sequencing (NEB Ultra II, New England Biolabs) as per the manufacturer's suggestions. Amplified libraries were quantified by high sensitivity fluorometry (DeNovix) and high sensitivity D1000 tape analysis using a 4200 TapeStation (Agilent Technologies).
attributes.tag: GEO Accession
attributes.value: GSM4678055