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Sample GSM3690736

Query DataSets for GSM3690736

Status

Public on May 05, 2020

Title

NSC_06_ATAC_Seq

Sample type

SRA

Source name

Neural Progenitor Cell

Organism

Homo sapiens

Characteristics

cell type: iPS-derived Neural Progenitor Cell

Treatment protocol

NPC are prepared using PSC Neural Induction Medium (Thermofisher) following vendor’s protocol with modification. Briefly, hiPSCs were replated as clumps in mTeSR media (STEMCELL) supplemented with 5 µM ROCK inhibitor (R&D Systems) on Day 0. From Day 1 to Day 9, cells were treated in PSC Neural Induction Medium with routinely removal of differentiated non-neuron cells. PSC Neural Induction Medium was prepared as 490ml Neurobasal Medium (Thermofisher) and 10 ml Neural Induction Supplement (Thermofisher). On Day 10, P0 NPCs were ready for passaging. From P0 to P3, NPCs were passaged using Rosette Selection Reagent (STEMCELL) and expanded in Neural Expansion Medium. Neural Expansion Medium was prepared as 245 ml Neurobasal Medium, 245 ml Advanced DMEM⁄F-12 media (Thermofisher) and 10ml Neural Induction Supplement. From P4, NPCs were passaged as single cells using Accutase (Thermofisher). 5 µM ROCK inhibitor were supplemented into media on the day of passage and withdrawn 24 hr post replating.

Extracted molecule

genomic DNA

Extraction protocol

For ATAC-seq, we harvested 50,000 cells by centrifugation at 500 g x 5 min at 4C. We washed cells once with PBS buffer and resuspended the cell pellets in 50 uL of cold lysis buffer (10 mM Tris-HCl, pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA- 630). We collected cell pellets, discarded the supernatants, and immediately continued to transposition reaction in 50 uL transposition reaction mix. We incubated the transposition reaction at 37C for 30 min, immediately followed by purification through a QIAGEN MinElute Kit. We stored the purified DNAs at 20 C until generating sequencing library.
The library was generated and sequenced at University of Minnesota Genomic Center.We have followed the protocol as previously described (Buenrostro et al., 2013) with minor modifications. Specifically, we thawed the transposed and purified DNAs and carried out PCR in 50 uL of reaction with NEBNext High Fidelity 2X Master mix and 5 ul of 25 mM Forward/Reverse ATAC-seq index, i.e., standard barcoded primers of Nextera kit (FC-121-1030), for each sample. After reaction, we purified samples with SPRI beads and eluted the PCR products in 12 uL elution buffer. We checked the DNA library by picogreen quantification and the size of the fragments with an Agilent High Sensitivity DNA chip. Libraries were diluted to 2 nM and pooled for downstream sequencing.
The library was generated and sequenced at University of Minnesota Genomic Center.We have followed the protocol as previously described (Buenrostro et al., 2013) with minor modifications: (1) using the standard Nextera primer set for PCR amplification of the transposed DNA fragments, (2) monitoring qPCR reaction to determine the number of additional cycles for full PCR amplification, and (3) using Agilent High Sensitivity DNA chip for library size validation. Specifically, we thawed the transposed and purified DNAs and carried out an initial 5-cycle PCR (1 cycle: 72 C for 5 min; 1 cycle: 98 C for 30 s; then 5 cycles: 98 C for 10 s, 63 C for 30 s, 72 C for 1 min) in a 50 mL of reaction including NEBNext High Fidelity 2X Master mix and 5 mL of 25 mMForward/Reverse ATAC-seq index, i.e., standard barcoded primers of Nextera kit (FC-121-1030), for each sample.We then used qPCR to determine the number of additional cycles of PCR amplification that were required, by using the cycle number that corresponds to 1/3 of the maximum fluorescent intensity (typically +5, +6, +7) shown on real-time PCR plots on ABI7900 (Applied Biosystems). After the reactions were subjected to additional PCR cycles, we purified each sample with SPRI beads and eluted the PCR products in 12 mL elution buffer. We checked the DNA library by picogreen quantification and the size of the fragments with an Agilent High Sensitivity DNA chip (the library should be sized between 200-700 bp). Libraries were diluted to 2 nM and pooled for downstream sequencing. For the initial OCR profiling, we pooled all the 6 libraries (2 for iPSC, 1 for N-d27, 1 for N-d33, and 2 for N-d41) and sequenced on a HiSeq 2500 using a PE 2x50 bp flow cell in a single sequencing lane. For the additional ATAC-seq on the unedited and edited iNs, we followed the same procedures as described above. We achieved > 120 million passing-filter reads for the lane and the average quality scores for all libraries are all above Q30 for both R1 and R2.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Data processing

ATAC-Seq data: All raw sequence reads generated by Illumina HiSeq 2000 have been de-complexed at the University of Minnesota Genomic Center and provided as 2x50 bp pair-end fastq files (100 M reads per sample). Adapter remnants, low-quality reads, and low QSEQ short sequences near either end of reads were processed by Trimmomatic (ILLUMINACLIP:NexteraPE-PE.fa:2:30:7, SLIDINGWINDOW:3:18, MINLENGTH:26). The survived sequences were separated into pair-end and single-end fastq files per sample, respectively. The pair-end and single-end fastq files were individually mapped against the human genome reference file including decoy sequences (GRCh38p7/hg38, 1000 Genome Project) using bowtie2 (-x 2000, -mm --qc-filter –met 1 –sensitive –no-mixed -t) and subsequently merged and sorted as BAM-formatted files using samtools, only uniquely mapped reads (MAPQ > 30) were collected. Picard tools MarkDuplicate was then used to remove all PCR and optical duplicated reads from the BAM file.
RNA-Seq data: RNA sequencing files were provided in the format of 2x150 bp pair-end (25 M reads per sample) fastq files. Briefly, libraries were prepared using the NEB Nextera kit with customised adapters. Since the sequencing facility had performed pre-cleaning on raw reads, no Trimmomatic action was performed. The files were subsequently mapped to the human hg38 genome (GRCh38p7) using STAR v 2.6.0 (--outSAMtype BAM SortedByCoordinate --quantMode GeneCounts --outSAMattrIHstart 0 --outSAMstrandField intronMotif --outSAMmultNmax 1 --outFilterIntronMotifs RemoveNoncanonical --outBAMcompression 10 --outBAMsortingThreadN 20 --outBAMsortingBinsN 20 --outFilterMultimapNmax 1 --outFilterMismatchNmax 1 --outSJfilterReads Unique --limitBAMsortRAM 10000000000 --alignSoftClipAtReferenceEnds No --quantTranscriptomeBAMcompression 10 10 ).
Genome_build: GRCh38.p7

Submission date

Mar 28, 2019

Last update date

May 05, 2020

Contact name

Jubao Duan

E-mail(s)

[email protected]

Phone

(224) 364-7564

Organization name

NorthShore University HealthSystem