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Sample GSM8162306

Query DataSets for GSM8162306

Status

Public on Jun 30, 2024

Title

HCT_mRNA_Med26Degron_IAA_r1_4h

Sample type

SRA

Source name

HCT116

Organism

Homo sapiens

Characteristics

cell line: HCT116
cell type: colorectal carcinoma cell
genotype: Med26-degron
treatment: 10% FBS 14h, noFBS+DMSO 6h, FBS + DMSO2h, 500uM IAA 12h, FBS+actinomycin D 4h

Treatment protocol

HCT-116 degron cell lines: Starvation: cells were FBS depleted for 14 hours, followed by an additional incubation with FBS free medium for another 6 hours in the presence of DMSO (control), 500 µM auxin (indole-3-acetic acid, IAA) (Sigma-Aldrich) for degradation of the AID-tagged RAD21 and MED26 or 500nM JQ-1 (Sigma-Aldrich) for BRD4 displacement. Cells were analyzed after addition of FBS in the presence of DMSO or drugs at timepoints: 0h (RAD21 degron), 0.5h (RAD21 degron) and 2h (RAD21 and MED26 degron, JQ-1 treatment). ES (E14) For the AID-tagged proteins 500 µM auxin (indole-3-acetic acid, IAA) (Sigma-Aldrich) was added for 6 hours (RAD21) or 24 hours (CTCF). Splenic B cells To measure mRNA half-lives in activated B cells Actinomycin D was added after 2 days of stimulation with LPS, IL4 and anti-CD180 (more details: mRNA half-lives estimation). Skin cells To estimate mRNA half-lives in skin cells we used basal cells.

Growth protocol

HCT-116 degron cell lines: RAD21- mAID-mClover1 and HCT116-OsTIR1-MED26-AID2 were cultured in McCoy's 5A medium supplemented with L-Glutamine (Gibco), 10% FBS (Gemini), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), at 37 °C; 5% CO2. ES (E14) RAD213 and CTCF degron4 cell lines were cultured in knock-out DMEM (Gibco), ES cell qualified FBS (ATCC), 1:100 Glutamax (Gibco), 1:100 sodium pyruvate (Gibco), 1:100 NEAA (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), 1:1000 2-Mercaptoethanol (Gibco) and 1:10,000LIF (10,000x) on 0.1% gelatin coated dishes at 37 °C; 5% CO2. Primary WT and Myc knockout B cells were derived from Myc flox/flox mice (WT) or RosaCreERTam/Myc flox/flox mice (KO) were treated with tamoxifen (Sigma) (three times of i.p. 1mg/mouse at 4, 3 and 1 day before sacrifice). The deletion of floxed allele was checked as previously5 with PCR or intracellular staining of Myc protein. B cells were isolated from spleen of WT and KO mice, enriched with EasySep Mouse B Cell Isolation KIT (STEMCELL Technologies) and activated for 4 hours with 50 mg/ml LPS and 2.5 ng/ml IL4 in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gemini), 1:100 Glutamax (Gibco), 1:100 sodium pyruvate (Gibco), 1:100 NEAA (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), 1:1000 2-Mercaptoethanol (Gibco) and HEPES (Gibco), at 37 °C; 5% CO2 B220 positive primary bone marrow B cells were derived from C57BL/6J wild-type mice, enriched with CD45R (B220) MicroBeads Kit (Miltenyi Biotec) and Dead Cell Removal Kit (Miltenyi Biotec). Splenic B cells were derived from C57BL/6J wild-type mice immunized with a keyhole limpet haemocyanin (KLH; 25 µg/footpad, Sigma-Aldrich) in the presence of 10% CFA adjuvant (Sigma-Aldrich). After 5 days B cells were isolated from spleen, enriched with EasySep Mouse B Cell Isolation KIT (STEMCELL Technologies), and activated for 72 hours with 50 mg/ml LPS, 2.5 ng/ml IL4 and anti-CD180 in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gemini), 1:100 Glutamax (Gibco), 1:100 sodium pyruvate (Gibco), 1:100 NEAA (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), 1:1000 2-Mercaptoethanol (Gibco) and HEPES (Gibco), at 37 °C; 5% CO2. CH12 B lymphoma cells were cultured in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gemini), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), 1:1000 2-Mercaptoethanol (Gibco), at 37 °C; 5% CO2. LNK (NK cell line) cells were cultured in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gemini), 1:100 Glutamax (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), 10 mM HEPES, 1:100 NEAA (Gibco), 1:100 sodium pyruvate (Gibco), 1:1000 2-Mercaptoethanol (Gibco) and 200 IU/ml IL-2 (Sigma-Aldrich). Skin cells for scRNA-Seq: whole epidermis from mouse neonates. Keratinocytes were isolated from Fvb neonate (P0-P2) skin (n = 2) as described in Lichti et al., 20086. Cells were subjected to MACS Dead Cell removal kit (Miltenyi Biotech) using the manufacturer’s protocol. Skin cells for mRNA half-lives estimation: primary keratinocyte. Skins from a pool of Fvb neonatal mice were collected at P0-P2 and incubated overnight in 2.0 U/mL Dispase II (Sigma) in KBM-Gold (Lonza) on an end-over-end rotator. The following day, skins were washed twice in 1x PBS (Sigma), and epidermises were separated from dermises. Epidermises were placed basal side down and floated in pre-warmed 0.25% Trypsin-EDTA (Thermo Scientific) for 20min on an orbital shaker at room temperature. An equal volume of pre-warmed 2.5mg/mL Soybean Trypsin Inhibitor (Thermo Scientific) was added to finish trypsinization, and epidermises were rubbed vigorously against bottom of plate using curved forceps to dislodge keratinocytes. Dislodged cells were pooled into a tube on ice in KBM-Gold. Pooled cells were pipetted ~40 times with a 10ml serological pipette and strained with a 100um filter before pelleting cells at 300xg for 10min at 4°C. Cells were resuspended in 4.5mL KBM-Gold per epidermis and cell count was obtained using Cellometer ViaStain AOPI Solution (Sigma). The cells were subjected to Percoll (GE LifeSciences; ratio of 10X PBS:Percoll:KBM-Gold media was 1:4:5, where the cells were at a concentration of 1 million per mL of final volume) gradient ultracentrifugation using a swinging bucket rotor (11,000 rpm for 42 minutes at 4°C) to separate the basal and suprabasal fractions. The cells at the top band were suprabasal cells and separated from the pellet at the bottom of the tube which were basal cells. The basal cell sample was carefully pipetted out, washed twice with 4°C 1x PBS, seeded onto collagen-coated (Collage Type I; Corning) 6-well plates at 1x106 cells per well, and incubated at 37°C overnight to allow cells to attach to the plate before proceeding with experiments. Mast cells: Mouse bone marrow-derived mast cells (BMMC) were differentiated from the marrow of tibias and femurs of C57/BL6 mice and cultured for 8 weeks in RPMI 1640 containing 10% FBS, 1M HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, 4 mM l-glutamine, 1 mM sodium pyruvate, non-essential amino acids, 50 μM 2-mercaptoethanol, IL-3 (20 ng/mL) and stem cell factor (SCF) (20 ng/mL). The purity of mast cells was monitored by assessing the expression of the receptors for SCF (CD117;Kit) and for IgE (FcεRI) by flow cytometry. By the end of 6-8 weeks in culture more than 98% of the cultured population was FcεRI+/CD117+ double-positive mast cells.

Extracted molecule

total RNA

Extraction protocol

RNA-Seq
NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) was used for library construction. Single 50 cycles of sequencing data were acquired on NextSeq2000, NextSeq550, HiSeq3000 or NovaSeq6000 (Illumina).
ChIP-Seq
Library was prepared using Ovation Ultralow System V2 (Tecan) according to manufacturer’s protocol. Before sequencing high quality was confirmed using the Agilent TapeStation system. Single 50 cycles of sequencing data were acquired on NextSeq550, NovaSeq6000 (Illumina).
scRNA-Seq
Libraries were prepared using Chromium Single Cell 3′ Reagent Kits (10X Genomics) according to the manufacturer’s protocol. With double 10bp index cycles, 28 forward and 90 reverse cycles were run on NovaSeq6000 machine (Illumina).
RNA-Seq
NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) was used for library construction. Single 50 cycles of sequencing data were acquired on NextSeq2000, NextSeq550, HiSeq3000 or NovaSeq6000 (Illumina).
ChIP-Seq
Library was prepared using Ovation Ultralow System V2 (Tecan) according to manufacturer’s protocol. Before sequencing high quality was confirmed using the Agilent TapeStation system. Single 50 cycles of sequencing data were acquired on NextSeq550, NovaSeq6000 (Illumina).
scRNA-Seq
Libraries were prepared using Chromium Single Cell 3′ Reagent Kits (10X Genomics) according to the manufacturer’s protocol. With double 10bp index cycles, 28 forward and 90 reverse cycles were run on NovaSeq6000 machine (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq X Plus

Data processing

scRNAseq: The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v3.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
base calling: illumina CASAVA 1.8.2
Proseq_density: trim: cutadapt 1.14 for adapter and UMI removal with -O 1 --match-read-wildcards -m {20,26}), seqtk trimfq for R1l
Proseq_density: mapping: bowtie2 on combined genome (dm6 (spikein)+ hg19(primary))
Proseq_density: dedup: umitools (http://github.com/CGATOxford/UMI-tools)
Proseq_density: reads seperation with samtools 1.3.1 and convert to bedGraph through bowtie2stdBedgraph.pl (https://github.com/AdelmanLab/NIH_scripts)
mRNA-seq half-life estimation: alignment: gsnap --use-sarray=0 --novelsplicing=0 --use-splicing refseq.splices.iit --format=sam -B 4
mRNA-seq half-life estimation: htseq count file generation from mRNA-seq aligned both for ERCC spike-in control and genomic sequence
mRNA-seq half-lfe estimation:estimate the decay rate constant (kdecay) by fitting the exponential curve on concentration changes upon timepoints using SSasymp R function. From the decay constant, we calculated the mRNA half-lives using the following equation: ln(2)/kdecay
Assembly: hg19
Supplementary files format and content: Tab-separated values files and matrix files
Supplementary files format and content: bigwig file

Submission date

Mar 22, 2024

Last update date

Jun 30, 2024

Contact name

Seolkyoung Jung

Organization name

NIH

Department

NIAMS

Lab

biodata mining and discovery section