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| Status |
Public on Dec 13, 2023 |
| Title |
Human Female NHDF R1 – XIST Pulldown |
| Sample type |
SRA |
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| Source name |
NHDF
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| Organism |
Homo sapiens |
| Characteristics |
cell line: NHDF
cell type: Somatic
library strategy: RAP-Seq
experiment type: XIST RAP
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| Treatment protocol |
In addition to growing each cell type in appropriate media condition, cells were not treated with any additional agent(s).
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| Growth protocol |
Naïve hESCs (UCLA1 and H9) and iPSCs were cultured in 5iLAF naïve ESC medium on irradiated male mouse embryonic fibroblasts (1:1 mixture of DMEM/F12 and Neurobasal supplemented with N2, B27, penicillin/streptomycin, nonessential amino acids, GlutaMAX, 0.5% KSR, 0.1mM β-mercaptoethanol, 50ug/ml bovine serum albumin, 20ng/ml rhLIF, 20ng/ml Activin A, 8ng/ml FGF2, 1uM MEK inhibitor PD0325901 0.5uM B-Raf inhibitor SB590885, 1uM GSK3-β inhibitor IM-12, 1uM Src inhibitor WH-4-023, and 10uM ROCK inhibitor Y-27632). NHDFs were cultured in DMEM, with 10% FBS, penicillin/streptomycin, nonessential amino acids, GlutaMAX and 0.1mM β-mercaptoethanol. H9 hESCs were cultured in 5% CO2, 5% O2 (Hypoxia) at 37C and UCLA1, iPSCs and NHDFs were cultured in normal atmospheric O2 and 5% CO2 at 37C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
After washing with PBS, 5-30 million cells were harvested from confluent cell cultures and incubated with freshly-made 10ml 2mM DSG in 1x PBS at room temperature for 45 minutes. Cells were subsequently crosslinked further at room temperature with 10ml 3% formaldehyde for 10 minutes, and the reaction was stopped by the addition of 2ml 2.5M glycine. Cells were pelleted at 4˚C, aliquoted into vials of 10 million cells, and subjected to lysate preparation as described by Engreitz et al. (Engreitz et al., 2013). Briefly, the cells were passively lysed in Cell Lysis Buffer (10mM HEPES at pH7.5, 20mM KCl, 2mM MgCl2, 1mM EDTA with 0.1% NP-40) for 10min on ice, then lysis was completed either with glass dounce homogenizer (UCLA1, naïve iPSCs and NHDFs) or without (naïve (WT and with XIST KO) and primed H9, and primed iPSCs). Nuclei were lysed in Nuclear Lysis Buffer (20mM HEPES at pH7.5, 50mM KCl, 1.5mM MnCl2, 1% NP-40, 0.4% Sodium deoxycholate, 0.1% N-lauroylsarcosine) for 10min on ice. Chromatin was solubilized by sonication. Briefly, for UCLA1, naïve iPSCs and NHDFs, five cycles of sonication were performed, with each cycle lasting 45 seconds on high power. For naïve and primed H9, and primed iPSCs sonication was done with a Misonix S-400 sonicator with microtip (model number: U1240A0418) at an amplitude of 12, total process time of 2 min with pulse on for 1sec and pulse off for 3sec. After sonication, chromatin was further segmented by incubating it with TURBO DNase digestion at a concentration of 0.1-0.4U/ul for 12-20min at 37C (time was optimized for each sample). The digestion was stop by the addition of DNase Stop Solution (mixture of EDTA and EGTA). XIST RNA pulldown from 5 million cells was done using 1ug (UCLA1, iPSCs, NHDF R1) or 50pmol (primed and naïve H9 and primed iPSCs) of non-overlapping 90nt long biotinylated oligonucleotide probes (Eurofins) (Table S3). To test the effect of higher concentration of the biotinylated oligonucleotides, a higher concentration was added to one replicate of NHDF (R2), using 5ug probes. 1mg (UCLA1, naïve iPSCs and NHDFs) or 0.6mg (naïve and primed H9, and primed iPSCs) of C1 Streptavidin beads were used. As a control, 5% of each sample was taken as an input control and the remaining sample was used for the pulldown. DNA was eluted by Rnase H digestion, and crosslinking was reversed via proteinase K digestion of eluted DNA at 60˚C 10-12 hours.
DNA libraries were prepared using NEBNext Ultra End Rpair/dA-Tailing Module (NEB) and TruSeq DNA adapters (Illumina) ligated using Quick Ligase (NEB). Libraries were amplified by KAPA HiFi Polymerase (Roche), pooled, and sequenced on the Illlumina HiSeq platform to generate 50bp single-end reads (UCLA1, naïve iPSCs and NHDFs) or pair-end reads (naïve and primed H9 and primed iPSCs).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
RAP-Seq using probes targeting Xist in NHDFs (Rep1)
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| Data processing |
Trim: trim_galore (v0.4.1) with default parameters was used to trim DNA sequencing reads
Align: Bowtie2 (v2.2.9) with the default parameters was used to align reads
Filter: Samtools (v1.9) was used to remove reads with mapping quality less than 30, or marked by Picard MarkDuplicates (v2.1.0) as PCR duplicates
Count: Bedtools (v2.26.0) intersect was used to count reads in each genomic region (either 100kb windows every 25kb, 1Mb every 250kb, or around transcription start site)
Enrichment: read counts in each window were normalized by the total read counts in each sample. The ratio of the normalized reads in the pulldown to the input were used as inputs
Peaks: MACS2 (v2.2.7.1) using broad and max-gap=100 was used to call peaks
Assembly: hg38
Supplementary files format and content: bedgraph files represent normalized enrichment ratios in windows across the genome (either 100kb windows every 25kb, 1Mb every 250kb, or around gene TSS). Is_unmappable=yes mark unmappable genomic regions
Supplementary files format and content: peak files represent broad peaks called for XIST pulldown files samples compared to input
Library strategy: RAP-seq
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| Submission date |
Aug 22, 2023 |
| Last update date |
Dec 13, 2023 |
| Contact name |
Kathrin Plath |
| Organization name |
UCLA
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| Street address |
BOX 951737, 36-133 CHS
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095-1737 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE241439 |
XIST directly regulates X-linked and autosomal genes in naïve human pluripotent cells [RAP-seq_human] |
| GSE241444 |
XIST directly regulates X-linked and autosomal genes in naïve human pluripotent cells |
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| Relations |
| BioSample |
SAMN37116355 |
| SRA |
SRX21457663 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7729014_Autosomal_NHDF_1_1000.broadPeak.gz |
1.9 Kb |
(ftp)(http) |
BROADPEAK |
| GSM7729014_XIST_vs_Input_NHDF_1_100kb_25kb.bedgraph.gz |
645.6 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM7729014_XIST_vs_Input_NHDF_1_1Mb_250kb.bedgraph.gz |
56.2 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM7729014_XIST_vs_Input_NHDF_1_TSS_50kb.bedgraph.gz |
560.2 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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