|
| Status |
Public on Jun 07, 2023 |
| Title |
A99_IgG_CUT |
| Sample type |
SRA |
| |
|
| Source name |
A99
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: GIS-NEC (ASCL1 positive)
antibody catalog/vendor: PP64B, Merck Millipore
antibody: IgG
|
| Treatment protocol |
CUT&Tag: 100,000 cells were prepared and bound to concanavalin-coated magnetic beads. After permeabilizing the cells with digitonin, a primary antibody was added and incubated at room temperature for 2 hours. Next, a secondary antibody was added and incubated at room temperature for 1 hour. The beads were washed and CUTANA pAG-Tn5 for CUT&Tag was bound to the secondary antibody. After incubated with a solution containing Mg2+ to cleave the target DNA, the DNA was extracted and amplified by PCR. The PCR product was purified using AMPureXP magnetic beads. Sequence libraries were pooled at 5 nM.
|
| Growth protocol |
ATAC-seq: 50,000 nuclei were prepared for each cell and incubated with transposition buffer containing Tagment DNA Enzyme1 and Tagment DNA Buffer. The transposed fragment was amplified by PCR and the PCR product was purified using AMPure XP beads. The sequence library was pooled at 5 nM.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq: 5.0 × 107 fixed cells were lysed to prepare nuclear extracts. Chromatin was sheared by sonication and the lysates were incubated overnight at 4°C with Dynabeads protein A coupled with anti-H3K4me3 and anti-H3K27ac antibody. After immunoprecipitation, beads were recovered using a magnet and washed; next, chromatin was eluted, and cross-links were reverted overnight at 65°C. DNA was purified and quantified with the Agilent Bioanalyzer.
RNA-seq: Total RNA was extracted using a RNeasy Mini kit. Libraries were prepared using TruSeq stranded mRNA Sample Preparation Kit according to manufacturer's protocol.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Library strategy: CUT&Tag
ATAC-seq: Reads were aligned against GRCh38 by Bowtie2 (version 2.3.5.1) with the following parameters: --very-sensitive --local --no-mixed --no-discordant -I 10 -X 2000. Duplication reads were removed by picard MarkDuplicates (version 2.23.8) and peak calling was performed with Genrich (version 0.6, available at https://github.com/jsh58/Genrich) with default parameters.
CUT&Tag: Quality control, alignment and removal of duplication reads was performed using the same pipeline with ATAC-seq. Peak calling was performed by SEACR (version 1.4) for H3K27ac (relaxed mode with using non-normalized IgG control track), H3K4me3 (stringent mode with using non-normalized IgG control track) and ELF3 (stringent mode without IgG control track with following parameter: -c 0.01)) and SICER2 (version 1.0.3) for H3K27me3.
ChIP-seq: Reads were mapped to GRCh38 using Bowtie2. Peak calling was performed using MACS2 with the following parameters: -q 0.0001. The input DNA sequence was used as a control.
RNA-seq: STAR (version 2.7.0f) was used for alignment against GRCh38 and read counts were calculated by RSEM (version 1.2.28) with TPM normalization.
Genome_build: GRCh38
Supplementary_files_format_and_content: bed
Supplementary_files_format_and_content: gene.result
|
| |
|
| Submission date |
Dec 10, 2021 |
| Last update date |
Jun 07, 2023 |
| Contact name |
Masafumi Horie |
| E-mail(s) |
mhorie-tky@umin.ac.jp
|
| Organization name |
University of Southern California
|
| Street address |
2011 Zonal Ave
|
| City |
Los Angeles |
| ZIP/Postal code |
90033 |
| Country |
USA |
| |
|
| Platform ID |
GPL20795 |
| Series (1) |
| GSE190618 |
The role of ELF3 in GIS-NEC and SCLC |
|
| Relations |
| BioSample |
SAMN23845321 |
| SRA |
SRX13373942 |
