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Sample GSM8059867

Query DataSets for GSM8059867

Status

Public on May 13, 2024

Title

patient_MPRA_rep1

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

An oligo pool containing 238bp sequences centered on each of the 1,693 rare, recessive variants identified in HARs, VEs, or CNEs in the HMCA cohort and in HARs or VEs in the NIMH cohort with or without the variant of interest was synthesized by Twist Biosciences. Each sequence was represented 10 times in the oligo pool with different 12-mer barcodes. The oligo pool was amplified with PCR primers to add SfiI restriction sites, and then cloned and tested 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. 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 72 hr post-transfection. Six replicates were performed.

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

Illumina HiSeq 2000

Description

patient_MPRA_results.xlsx

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).
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 6 cDNA replicate pools were used in the analysis. Enhancer activity was assessed with the wilcoxon test at 5% FDR.
Assembly: hg19
Supplementary files format and content: xlsx file of patient MPRA results for HARs, VEs, and CNEs
Library strategy: MPRA

Submission date

Feb 02, 2024

Last update date

May 13, 2024

Contact name

Janet Song

Organization name

Boston Children's Hospital

Lab

Walsh

Street address

3 Blackfan St