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Target of Dleu2-17aa in CD4+ T cells

ABSTRACT: To identify Dleu2-17aa micropeptide interacting proteins in CD4+ T cells, we performed pull-down assays fllowed by LC-MS using biotin-labeled Dleu2-17aa. As controls, we included the unlabeled free Dleu2-17aa and a biotin-only group. Pull-downs were carried out in both primary mouse CD4+ T cells and the EL4 T cell line.

INSTRUMENT(S): 6520A Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Primary Cell, T Cell

SUBMITTER: Sibei Tang

LAB HEAD: Honglin WANG

PROVIDER: PXD046468 | Pride | 2023-12-23

REPOSITORIES: Pride

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Project description:2×10^7 HL60 cells were treated with 10 uM ATRA for 2 and 5 days, and conducted Hi-C to study the chromosomal architecture. Cells were fixed by 1% formaldehyde (v/v) for 15 min at room temperature (RT) with slowly rotation. Cells were lysed by 550 μl lysis buffer (500 μl 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and 50 μl protease inhibitors) using a homogenizer, and harvested the chromatin at 5,000 rpm and washed twice. The pellet was added 1% SDS and incubated at 65 oC for 10 min, then quenched by Triton X-100. The chromatin was digested by 600 U MboI (NEB, Ipswich, USA) at 37 oC overnight. The next day, enzymes were inactivated by 10% SDS at 65 oC for 30 min, and the restriction fragment overhangs were filled in using biotin-14-dCTP and Klenow at 37 oC for 45 min, and the ligation was conducted in 7.61 ml ligation mix (745 μl 10% Triton X-100, 745 μl 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT), 80 μl 10 mg/ml BSA, 80 μl 100 mM ATP and 5.96 ml water) for 4 h at 16°C. The blunt-end Hi-C ligation was sonicated and pull down by streptavidin beads, then purified by ethanol. Hi-C library was prepared for paired ending sequencing using NEBNext® UltraTM DNA Library Prep Kit for Illuina® (NEB). For regular 3C ligation, after enzymes inactivation, 10 μl 1 U/μl T4 DNA ligase was added to incubate at 16°C overnight. The interaction was detected by qPCR using the specific 3C primers (Table 1) followed by DNA purification. Hi-C was conducted in PE-150. An iterative method for Hi-C mapping with initial length 30bp, if it fails to map uniquely, the next round of mapping for additional 20 bp continued, this procedure lasts until the full read length reaches 150bp. The Hi-C reads were mapped to the human reference genome (assembly GRCh38) using bowtie2 (v2.2.9). The reads mapped to the same restriction fragment, the reads less than 500bp and PCR duplicates were all removed. Hi-C contact maps were normalized using ICE.

			Action	DRS
	WHL_1.raw	Raw
	WHL_2.raw	Raw
	WHL_20200122_PEAKS_8.zip	Other
	WHL_3.raw	Raw
	proteinSeq.txt	Txt

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Target of Dleu2-17aa in CD4+ T cells

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