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Sample GSM4577356

Query DataSets for GSM4577356

Status

Public on Jul 23, 2020

Title

853T2_Cas9_Control_rep1

Sample type

SRA

Source name

853T2; tumor-derived lung cell line

Organism

Mus musculus

Characteristics

strain: mixed; C57/BL/6J; 129/SV
tumor stage: normal; 25 weeks post-initiation
cell line: 853T2
treatment: Cas9_Control

Treatment protocol

Tumor were induced at age 6-10 weeks. Mice were provided Adeno-SPC-Cre virus intratracheally resulting in recombination of Kras G12D, p53, and tdTomato in alveolar type II cells. Cell lines were generated followed by CRISPR perturbation.

Extracted molecule

genomic DNA

Extraction protocol

Individual tumors were dissected, digested in an enzymatic buffer (1X HBSS, 5mM HEPES, DNaseI, Collagenase IV), and incubated with rotation at 37°C for 30 minutes. The enzymatic buffer was quenched with DMEM and spun at 1000 rpm. Cell pellets were resuspended in DMEM and plated in 6-well plates to allow for attachment. Cell lines were genotyped for Kras, p53, and tomato after 5 passages in culture. The cell lines used in this study were established from mouse LUAD over the course of the study. All lines were grown in DMEM, 10% FBS, and 1% pen-strep. KP cell lines have not been authenticated because the cell lines are not found in established databases. The KP cell lines were tested for mycoplasma and found to be negative. GM12878 cells were grown in DMEM, 10% FBS, and 1% pen-strep and 3T3 cells were grown in RPMI 1640, 10% FBS, and 1% pen-strep. GM12878 cells were authenticated by STR Profiling Service from ATCC.
For CRISPR knockout experiments, guides were cloned into the lentiCRISPR-V2 lentiviral vector (Joung et al., 2017). The lentiCRISPR-V2 vector was digested with Fast Digest EspI and ligated with EspI-compatible annealed oligonucleotides for sgRNAs. KP cell lines were infected with constructs containing guides and selected with puromycin after 48 hours. After puromycin selection, guide performance was tested by western blotting. For CRISPR activation (CRISPRa) experiments, the Lenti-Sam-puro construct was used (developed in the Jacks lab), an adaption of the previously published Lenti-Sam activation construct (Pentinmikko et al., 2019). EspI-compatible cloning was completed and cells were infected with constructs containing guides based on CRISPRa prediction software. Truncated guides of 15 bp were cloned into a lentiviral based expression construct which also encodes for a transcriptional activation complex (MS2-P65-HSF1) and a puromycin selection cassette. Non-metastatic and metastatic cell lines were engineered to express Cas9 following stable selection of a Cas9-Blast construct.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Data processing

Base calls were converted to fastq format using bcl2fastq. Raw sequencing reads were trimmed using custom python scripts to remove adapter sequences. The reads were aligned to hg19 or mm10 genome using Bowtie2 (Langmead et al. 2012) with maximum fragment length set to 2 kb, and all other default settings (bowtie2 -X2000 --rg-id). The data were demultiplexed tolerating one mismatched base within barcodes. Mitochondrial, discordant and low quality reads were removed using SAMtools (Li et al. 2009) (samtools view -b -q 30 -f 0x2). Duplicate sequences were removed using the picard toolkit (http:// broadinstitute.github.io/picard/). Profiles for all cells were first merged into a single alignment (.bam) file and used as input for peak calling with MACS v2.1.2 (MACS2) (Zhang et al., 2008). All default options were used, with the following flags explicitly set: --nomodel, --nolambda, --keep-dup all, --call-summits. This returned a list of single base pair peak summits with associated significance scores (corresponding to log FDR q-value from MACS2). Only peak summits with FDR < 0.01 were retained. Next, a previously described iterative filtering approach was implemented to obtain a list of significant, non-overlapping fixed-width peak windows (Lareau et al., 2019). Briefly, the called peak summits were first padded with 150 base pairs (bp) at either end to generate evenly sized 301 bp window peak regions. Peaks were then sorted in decreasing order of their significance scores. Keeping the most significant peak, overlapping peak windows that had lower significance scores were identified and then removed. This was repeated for the next most significant peak window. Through this iterative process, lower significance overlapping peak regions were filtered out, resulting in 285,956 disjoint 301 bp peak windows.
Genome_build: mm10
Supplementary_files_format_and_content: count files are tab-delimited .txt files of reads in peaks across samples in sparse matrix format. Row names for peaks can be pulled from .bed peak file.
Supplementary_files_format_and_content: bam files for each individual sample are provided following peak calling and data processing
Supplementary_files_format_and_content: .bed peak file, can be used to find peaks for CRISPR data

Submission date

May 28, 2020

Last update date

Jul 23, 2020

Contact name

Lindsay M LaFave

E-mail(s)

lmlafave@mit.edu

Organization name

MIT

Department

Biology