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Sample GSM3937422

Query DataSets for GSM3937422

Status

Public on Jun 26, 2020

Title

iPSC-3 RNA-Seq

Sample type

SRA

Source name

Induced pluripotent stem cells

Organism

Homo sapiens

Characteristics

cell type: Induced pluripotent stem cells derived from CCD-1079Sk fibroblasts

Growth protocol

Fibroblasts were grown in DMEM medium supplemented with 10% FBS (Hyclone), Glutamax (Thermo Fisher Scientific) and NEAA (Thermo Fisher Scientific) and subcultured at a ratio of 1:3 when they reach 90% confluence. H1 cells were cultured on the hESC-qualified Matrigel (Corning, #354277) coated plates in mTeSR1 medium (StemCell Technologies, #05850). iPS cells were cultured on Matrigel with a daily change of E8 medium and passaged by collagenase. To generate iPSC-derived NPCs, detached iPSC colonies were suspended in embryoid body (EB) medium (DMEM:F12 with 20% KOSR, 2?M dorsomorphin and 2 ?M A 83-01) for 7 days. Floating EBs were attached on Matrigel and cultured in NPC medium (DMEM/F12, N2 supplement, NEAA and 2 uM cyclopamine). After 14 days, neural tube-like rosettes were mechanically picked and dissociated into single NPCs by Accutase. NPCs were maintained as monolayer in NPC medium with medium change every two days. For neuronal differentiation, NPCs were plated on Matrigel and cultured in Neurobasal medium supplemented with 2%B27, 2 mM L-glutamine, 10 ng/ml BDNF and 10 ng/ml GDNF.

Extracted molecule

total RNA

Extraction protocol

For Hi-C, nuclei were extraced after fixing using a cell lysis buffer. For ChIP-seq, nuclei were extracted and chromatins were fragmented by sonication. The TF/histone-DNA complexes were isolated by antibody. Total RNA was isolated from samples by RNeasy Mini Kit (Qiagen) according to the manufacturer?s instructions.
For eHi-C, briefly, the cells and tissues were first fixed with 1% formaldehyde and then quenched with 150mM glycine. The fixed samples were lysed using a cell lysis buffer or a tissue specific buffer. And nuclei were isolated and then digested with HindIII. After that, in situ proximity ligation was done by ligating the digested chromatin in a large volume. Ligation products were then reverse-linked with proteinase K and purified using phenol-chloroform. The purified DNA was then digested with DpnII and then self ligated using T4 DNA ligase. After purification, the DNA products were then re-linearized with HindIII followed by DNA purification. The resulting DNA were used for library construction. First, the DNA were end-repaired using End-it kit (Epicentre). Second, the end repaired DNA was A-tailed by Klenow fragment (3??5? exo?; NEB). Then, a customized Illumina truseq Adapter was ligated and the resulting DNA was then PCR amplified. The final libraries were quantified and sequenced on Illumina Hiseq platform.
All Hi-C libraries were constructed following illumina insctructions accompanying Truseq sample preparation kit. Random indexes were introduced for eHi-C libraries to remove PCR duplication. Generally, PCR amplification was done with 7-9 cycles. All ChIP-seq libraries were generated following a ChIPmentation protocol with Nextera adapters.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

RNAseq_Complete_geneList_EdgeR.xlsx

Data processing

The paired-end Hi-C reads were mapped to human genome hg19 using BOWTIE. Only first 36 bases were used for mapping when reads is longer. The two reads were mapped independently and then merged into pairs using in-house script. Duplicated read pairs from the same biological library were removed. Easy Hi-C reads were mapped to hg19 using BOWTIE. Because nearly all the mappable reads start with HindIII sequence AGCTT, we trimmed the first 5 bases from every read, took the next 36 bases, and added the 6-base sequence AAGCTT to the 5’ of every read before mapping using the whole 42 bases.
For Hi-C, we focus on cis-interactions and therefore only kept Hi-C paired-end reads which both ends are mapped to the same chromosome. Out of all the intra-chromosome paired-end reads, we also discard the reads with both ends mapped to the same HindIII fragments. Since cut-and-ligation events are expected to generate reads within 500bp upstream of HindIII cutting sites due to the size selection (“+” strand reads should be within 500bp upstream of a HindIII site, and “-“ strand reads should be within 500bp downstream a HindIII site), we only keep reads pairs with both ends satisfying this criteria. For eHi-C library, the only type of invalid cis- pairs are self-circles with two ends within the same HindIII fragment facing each other
We next split all these reads into three classes based on their strand orientations (“same-strand”, “inward”, or “outward”), and generated the resulting lists of fragment pairs (with Hi-C read counts) from each class of reads.
There are total ~840k fragments in human genome. They are assembled into ~335k anchors after short fragments (<5kb) are merged into neighboring anchors. For every anchor, we first count Hi-C/eHi-C reads from the anchor to every fragment within 2Mb range.
We estimated a background frequency between any two fragments based on the average reads count of all fragment pairs that have similar lengths, similar gap distance, GC content, and visibility. The fragment-to-fragment data can be then converted to anchor-to-anchor data by adding the read counts and background frequencies together based on the assignment of fragments to anchors. P values of the enrichment of Hi-C reads over the background frequency can be calculated using a negative binomial model. The statistical method has been described in previous publication (Jin, F. et al. Nature 2013, 503:290-294)
For RNAseq, basecalls/demultiplexing performed using Illumina CASAVA 1.8.2. Reads were aligned to Hg38 using STAR2.4.0. Read counting was done using HT-seq0.6.1 with Hg38 RefSeq as reference. Low count genes were filtered. Normalization by TMM. Differential expression analysis was done using EdgeR.
Genome_build: hg19
Supplementary_files_format_and_content: For each Hi-C and easy Hi-C data set, the fragment-to-fragment frequency are provided in the txt file. For each Chip-Seq replicate, peak-calling results are provided in the bed file.

Submission date

Jul 10, 2019

Last update date

Jun 26, 2020

Contact name

Xiaoxiao Liu

E-mail(s)

[email protected]

Phone

(216) 368-5293

Organization name

Case Western Reserve University