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Sample GSM1325328

Query DataSets for GSM1325328

Status

Public on Mar 22, 2014

Title

FIREWACh_BioRep1_Hae_TechRepC

Sample type

SRA

Source name

Mouse Embryonic Stem Cells

Organism

Mus musculus

Characteristics

cell line: E14TG2a
passages: 25-30
tag: 129/Ola

Treatment protocol

ESCs were transduced at a MOI of 7 (determined by p24 ELISA) to ensure that the maximum number of cells is transduced while favoring single copy integration. To increase the likelihood that any given NFR-lentiviral genome would be represented, the number of cells transduced was equivalent to more than ten times each library’s complexity. Thus 5x106 E14 ESCs were plated in complete ESC medium plus LIF in feeder free conditions on a 10 cm gelatin coated dish one day prior to transduction. The cells were then transduced overnight in 10 ml of complete ESC medium plus 8 ug/ml polybrene, containing 3.5x107 virus particles for a MOI of 7. The following day the medium was replaced with fresh ESC medium plus LIF. Hygromycin-selection was initiated four days post transduction in ESC medium/LIF containing 250 ug/ml hygromycin B. Cells were selected for hygromycin B resistance for 5 days, with media changed daily. GFP+ cells were selected using fluorescence activated cell sorting (FACS) on a iCyt Reflection HAPS2 cell sorter.

Growth protocol

E14ESCs were obtained from ATCC (ES-E14TG2a, CRL-1821) and were maintained in 2i/LIFwith the inhibitors CHIR99021 (3μM), and PD0325901 (1 μM) (Axon Medchem BV, The Netherlands) and 100 u/ml LIF in N2B27,or in standard ESC medium (DMEM supplemented with 15% FCS (Stem Cell Technologies,“ES cult”), 0.1mM non-essential amino acids, 0.1mM b mercaptoethanol, 1X Glutamax (Invitrogen), and 1000 u/ml LIFon plates coated with 0.2% Gelatin1. 3T3 were maintained in DMEM(Invitrogen), 15% Bovine Calf Serum and Penicillin/Streptomycin.

Extracted molecule

genomic DNA

Extraction protocol

NFR-DNAs were rescued from either the initial lentiviral plasmid libraries or gDNA of GFP+ selected cells using PCR in a method adapted from bacterial rRNA sequencing7. In this method, Illumina sequencing adaptors are included in the primers, permitting one step amplification and sequencing library preparation. Primers (termed FMS-F/R,) were designed such that they contain recognition sequences complimentary to lentiviral sequence flanking NFR-DNA and Illumina adaptor sequence for paired-end sequencing in addition to a 6base pair Index sequence. Six PCR reactions (10 ul Phusion Polymerase buffer, 1 μl 10 mMdNTP, 2.5 μl 10 μM forward and reverse primer, 1.5 μl DMSO, 0.5 μl (NEB) 50 ng DNA, 31 μl H20; 16 cycles with 550C annealing temperature) per plasmid library were pooled and sequenced. FIREWACh elements were recovered from the genomic DNA of a least 1x106 FACS-sorted GFP+ cells using PCR. In this case 10 PCR reactions were performed using the same conditions as above but 100 ng gDNA and 23 cycles of amplification. The 10 reactions were then pooled.
Six PCR reactions (10 ul Phusion Polymerase buffer, 1 μl 10 mMdNTP, 2.5 μl 10 μM forward and reverse primer, 1.5 μl DMSO, 0.5 μl (NEB) 50 ng DNA, 31 μl H20; 16 cycles with 550C annealing temperature) per plasmid library were pooled and sequenced. Quality was assessed determining size distrubution with with bioanalyzer and concentration was confirmed via QPCR
Up to six samples were pooled within a single lane on a MiSeq machine. Input library derived NFRs were sequenced together using three of the indices while FIREWACh NFRS, i.e. NFR’s rescued from GFP+ cells, were run with six samples per lane, each sample representing NFRs rescued from an independent biological replicate (i.e. HaeIII_BioRep1 etc). Technical replicates consisting of independent PCR rescued NFR sequencing libraries were sequenced on separate days.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Description

NFR DNA selected by GFP expression and rescued from the integrated lentiviral reporter within gDNA by PCR
FIREWACh.bed

Data processing

Illumina MiSeq 2x 151 bp data were pre-processed by demultiplexing and trimming of 7 bp from the 5' end and 44 bp from the 3' end, yielding a data set of 2 x 100 bp sequence reads.
Basecalls performed using Illumina's RTA software
Paired end sequences were aligned to the mouse reference genome (mm9) using BWA software with default settings.
Read pairs were filtered from the final data set if either read failed to map to the genome, if both reads did not map in the proper orientation, if the mapping quality score of both reads was less than 25, or if neither read had a unique map location on the genome. Target sites were identified as loci where paired reads both aligned entirely within a 500 bp genomic region.
Genome_build: mm9
Supplementary_files_format_and_content: .bed files containing the genomic loci of NFR elements within starting library (Library.bed) or elements rescued from sorted cells (FIREWACh.bed)

Submission date

Feb 11, 2014

Last update date

May 15, 2019

Contact name

Matthew Murtha

E-mail(s)

matthew.murtha@nyumc.org

Phone

2129929171

Organization name

NYU School of Medicine