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Sample GSM2213966

Query DataSets for GSM2213966

Status

Public on Sep 20, 2016

Title

D16, gDNA reads, Gal4-HP1, repl 1

Sample type

SRA

Source name

Kc167 cells

Organism

Drosophila melanogaster

Characteristics

cell line: Kc167
cell type: drosophila melanogaster dorsal closure stage embryonal cell line
transfected protein: Gal4DBD-HP1a

Treatment protocol

To establish the pool of cells harboring reporter constructs, 1 million Kc167 cells were transfected with 1 µg barcoded 5xGal4UAS-pMT-GFP plasmid library and 1 µg plasmid encoding Sleeping Beauty transposase (Addgene #65487) by electroporation. Transposase expression was induced by four heat shock treatments of 2.5 h at 37°C distributed over 36 h. Transfected cells were expanded until Sleeping Beauty expression was lost. For the resulting TRIP pool, we determined the number of integrations to be 0.3 per cell based on qPCR on gDNA for GFP integrations compared to a cell line with a single integration. A subpool of 30,000 cells was taken from the TRIP pool and expanded to limit the library complexity to a maximum of 10,000 integrations before gDNA was isolated for mapping of integrations. For assessing the effect of recruiting HP1a to integrated reporters, GFP expression was induced by adding 0.5 mM CuSO4 two days before the TRIP pool was transiently transfected with plasmids encoding either pAc5-Gal4DBD-V5-HP1a-T2A-mCherry or control plasmids encoding only Gal4DBD-V5 or V5-HP1a instead of Gal4DBD-V5-HP1. For assessing reporter expression by day two after transfection, cells were sorted for mCherry expression before gDNA and cDNA were isolated for further analysis. For determining reporter expression by day 16 after transfection, cells were kept unsorted before cDNA and gDNA isolation. To be able to compare bulk expression changes between cDNA samples, an independent TRIP pool with a complexity of approximately 200 barcodes was suspended in TRIsure (Bioline) and added to TRIP cells to an amount of 2.5% based on cell counts before RNA extraction.

Growth protocol

Kc167 cells were cultured at 23.5°C in Shields and Sang M3 Insect Medium (Sigma-Aldrich) with 0.25% Bacto Peptone (BD), 0.1% Yeast Extract (BD), 5% heat-inactivated FBS (Thermo Scientific) and 1% Penicillin/Streptomycin (Thermo Scientific). For pMT induction, sterile-filtered CuSO4 (Sigma-Aldrich) dissolved in H2O was added to 0.5mM final concentration

Extracted molecule

genomic DNA

Extraction protocol

To map the reporter integrations, gDNA was extracted from 20*106 cells using ISOLATE II Genomic DNA Kit (Bioline) and digested with NlaIII (40 U, NEB) in buffer 4 supplemented with BSA in a total volume of 100 µl for 2 h at 37°C. The reaction was terminated by heat inactivation for 20 min at 65°C. Fragments were circularized over night at 12°C using T4 Ligase (100 U, NEB) in T4 Ligase buffer in a total volume of 1600 µl. Products were precipitated with ethanol and purified with ISOLATE II PCR and Gel kit (Bioline). Unligated fragments were removed by digestion with Plasmid-Safe DNase (20 U, Epicentre) with added ATP (final concentration 1 mM) in the manufacturer-supplied reaction buffer in a total volume of 100 µl for 5 h at 37°C. The reaction was terminated by heat-inactivation for 30 min at 70°C and purified using ISOLATE II PCR and Gel kit (Bioline). To eliminate any remaining unintegrated plasmid , products were digested with I-CeuI (5 U, NEB) in buffer 4 supplemented with BSA in s total volume of 60 µl for 2 h at 37°C. The reaction was terminated by heat-inactivation for 20 min at 65°C and purified using ISOLATE II PCR and Gel kit (Bioline). Products containing barcodes and flaking gDNA were amplified in two rounds by inverse PCR to add Illumina adapters for Illumina paired-end sequencing. For cDNA reads, RNA from 10*106 transfected cells isolated by FACS sorting was extracted with Chloroform from TRIsure-resuspended samples (Bioline). The polyadenylated fraction was isolated using the Oligotex kit (Qiagen). RNA samples were treated with RNase-free DNase I (2 U, Roche) in DNase buffer in a total volume of 20 µl for 30 min at 37°C. The reaction was terminated by adding 1 µl of 25 mM EDTA and incubation at 70°C for 15 min. cDNA was generated using Tetro cDNA Synthesis Kit (Bioline) according to the manufacturer’s instructions with a GFP-specific primer instead of OligoDT. 1/4 of total cDNA was used in a PCR reaction to add adapters for Illumina single-end sequencing. The appropriate PCR cycle number was determined by qPCR to avoid overamplification. For gDNA reads, gDNA was isolated from 10*106 unsorted TRIP cells using ISOLATE II Genomic DNA Kit (Bioline). 500 ng were used in a PCR reaction to add adapters for Illumina single-end sequencing and amplified over 25 cycles.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

gDNA reads of Kc167 TRIP pool with 5xGal4UAS-pMT-GFP reporter integrations transfected with Gal4DBD-HP1a collected by sixteen days after transfection. Replicate 1 of 2
TRIP_HP1_D16

Data processing

Basecalling and filtering was performed using standard software of the illumina HiSeq 2500 or MiSeq. Extraction of 21 nt barcode reads from fastq files and alignment of TRIP mapping reads was performed using the TRIP script available at http://trip.nki.nl. We set a Hamming distance of 2 for removing mutated barcodes, a maximum distance of 500 nt on the forward read and 20 nt on the reverse read to cluster the positions during mapping and a minimum read number of 1. Spike-in library gDNA was sequenced separately to determine the spike-in barcode sequences. A list of the most abundant spike-in barcodes with over 1000 counts in gDNA was used for extracting spike-in reads from the TRIP samples. For each TRIP experiment sample, cDNA read counts per barcode were divided by the sum of cDNA read counts for all spike-in barcodes to correct for sequencing depth and standardize expression between samples. gDNA read counts were transformed to counts per million (cpm) to correct for sequencing depth. Spike-in corrected cDNA counts were then divided by gDNA cpm counts which yields the normalized expression per barcode. We set a cutoff for barcodes that had at least 100 gDNA reads in all samples to ensure that each barcode was sufficiently represented in the pool. To ensure reliable allocation of barcode integrations, we worked with a total of 1093 barcodes that fit the following criteria: (1) more than 2 reads in both forward and reverse mapping (2) 80% of forward and reverse mapping reads matched with the first mapping location and less than 10% of forward and reverse mapping reads matched with the second mapping location (3) mapq score of 10 or higher for forward and reverse mapping.
Genome_build: dm3
Supplementary_files_format_and_content: CSV files containing barcode sequences, position of integration, read counts and normalized expression for each of the samples
Supplementary_files_format_and_content: space delimited list of spike-in barcode sequences

Submission date

Jun 24, 2016

Last update date

May 15, 2019

Contact name

Bas van Steensel

E-mail(s)

b.v.steensel@nki.nl

Phone

+ 31 20 512 2040

Fax

+31 20 669 1383