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Sample GSM7790793

Query DataSets for GSM7790793

Status

Public on May 13, 2024

Title

HAR_caMPRA_D3_rep4

Sample type

SRA

Source name

Neuro2A/N2A cells (ATCC, CCL-131)

Organism

Homo sapiens

Characteristics

cell line: Neuro2A/N2A cells (ATCC, CCL-131)

Treatment protocol

Molecular Inversion Probes (MIPs) targeting HARs, VEs, and CNEs were designed using the MIPgen program (Boyle et al., 2014) and manufactured as a pool by CustomArray (Bothell, WA). Target genomic sequences were captured from DNA from sample NA12878 (Coriell Institute), and the caMPRA assay then was conducted as previously described (Girskis et al., 2021). Briefly, the captures were cloned into modified pMPRA1 vector (Addgene, 49349). The only modification to the pMPRA1 vector was the addition of an AsiSI restriction site used for inserting our captured DNA fragments. The luciferase gene and minimal promoter (pMPRA_donor2, Addgene, 49353) was then cloned into the pMPRA1_Capture construct pool. For random mutagenesis, we performed error-prone PCR using the GeneMorph II Random Mutagenesis Kit (Agilent, cat #200550) on the amplified, captured sequences. N2A cells were grown as adherent cultures in 15 cm plates. Then, 15ug of MPRA constructs were transfected into cells with Lipofectamine LTX reagent (Thermo Fisher, A12621). The cells were harvested 24 or 72 hr post-transfection. Five replicates were performed per condition.

Growth protocol

Neuro2A/N2A cells (ATCC, CCL-131) were grown in 10% fetal bovine serum, Dulbecco’s Modified Eagle Medium, and 1% Penicillin-Streptomycin in a 5% CO2 incubator at 37C.

Extracted molecule

polyA RNA

Extraction protocol

Cell pellets were washed with 1x PBS, and mRNA was extracted using the Dynabead mRNA Direct kit (Thermo Fisher, cat #61012), according to the manufacturer’s instructions.
mRNA was reverse transcribed using Superscript VILO Master Mix with EZ DNase (Thermo Fisher, cat #11766050). caMPRA barcodes were extracted using PCR amplification with primers containing illumina adapters for both the cDNA and plasmid pools and sent out for 150bp sequencing using Hiseq instruments at Psomagen (Rockville, MD).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

HiSeq X Ten

Data processing

Sequencing data from the plasmid DNA and cDNA libraries described above were processed with cutadapt to remove adapters (Martin, 2011).
Barcodes were extracted using UMI-tools (Smith et al., 2017)
Reads were mapped using bwa mem (Li and Durbin, 2009) and assigned to caMPRA probes using featureCounts (Liao et al., 2014).
The 10bp barcode were clustered to recover barcodes with small sequencing errors using the multiplexed version of the UMI-tools directional method.
Barcodes for plasmid and cDNA samples were normalized to a barcode per million format to remove bias due to sequencing coverage. Each cDNA barcode was normalized to the barcode count in the plasmid pool, and log2 transformed. Only barcodes that were found in the plasmid pool and all 5 cDNA replicate pools were used in the analysis. Enhancer activity was assessed with the XXXX test at 5% FDR.
Assembly: hg19
Supplementary files format and content: xlsx file of caMPRA results for HARs, VEs, and CNEs
Supplementary files format and content: xlsx file of random mutagenesis caMPRA results for HARs
Library strategy: MPRA

Submission date

Sep 19, 2023

Last update date

May 13, 2024

Contact name

Janet Song

Organization name

Boston Children's Hospital

Lab

Walsh

Street address

3 Blackfan St