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Sample GSM4890421 Query DataSets for GSM4890421
Status Public on Apr 29, 2021
Title 520 Spleen-1
Sample type SRA
 
Source name mouse mammary carcinoma cells implanted in mouse
Organism Mus musculus
Characteristics injected cell line: AT-3
metastic sites: Spleen
host: C57BL/6J
mouse id: 520
Growth protocol MDA-MB-231 FR and AT-3 FR cells were transduced with TLCV2-hgRNA A26 and selected by puromycin for 2 weeks. 1E5 barcoded cells were then implanted to the fourth pair of mammary fat pad of nude mice or C57BL/6 mice. 5 weeks later for MDA-MB-231 tumors or 18 days later for AT-3 tumors, the mammary tumors were resected and mice were give with a single dose of 5mg/kg doxycycline weekly for 5 cycles or 4 cycles through I.P. injection. 2 weeks after Dox treatment, the tissues with metastases were collected, and total DNA were exacted. For IIA injection model, 1E5 barcoded MDA-MB-231 cells were then delivered to bone via IIA. 2 weeks later, a single dose of 5mg/kg doxycycline weekly for 5 cycles was applied to the mice carrying bone metastases through I.P. injection. 3 weeks after Dox treatment, lung, right and left hindlimbhind limbs were collected, and total DNA were exacted. For in vitro treatment samples, cells were treated with 100μg/ml doxycycline for 2 hours and then rinsed by pre-warmed PBS twice to completely remove doxycycline and then allow to grow in vitro for 4 days. Then 1 million cells were collected for barcode sequencing and 0.5 million cells were seeded back to the petri dish and received another round of doxycycline treatment 24 hours later.
Extracted molecule genomic DNA
Extraction protocol The metastatic lesions were dissected with ex vivo BLI imaging, and the uninvolved tissues were removed. The surgery tools were sterilized with a bead-sterilizer following by 70% isopropanol between different lesions. The collected tissues was snap-frozen in the liquid nitrogen until DNA extraction. The tissues were then rinsed by the gDNA lysis buffer from Quick-DNA Miniprep Plus Kit (Zymo Research, D4068) and homogenized with Precellys Lysing Kit (Bertin Instruments, MK28-R). After the homogenization, samples were lysed at 55℃ for 3 h, followed by 0.33 mg/mL RNaseA treatment at 37℃ for 15 min. Nucleic acid was further extracted following the manufacturer’s instruction. The final DNA was assessed by NanoDrop 2000 (Thermo Scientific) and human/mouse DNA ratio was examined by q-PCR with primers targeting human and mouse GAPDH gene, respectively.
Barcodes were amplified by two rounds of PCR. The first round of PCR was performed with 100 ng genomic DNA using Platinum Taq DNA Polymerase (Invitrogen) with Barcode-For and Barcode-Rev primers in 15 cycles. The second round of PCR were performed in a real-time setting and stopped in mid-exponential phase using PowerUp SYBR Green Master Mix (Thermo Fisher) with Barcode-P5-For and Barcode-P7-Rev primers. PCR products were then column-purified with QIAquick PCR purification Kit (QIAGEN) and assessed with Qubit. The NEBNext Multiplex oligos for Illumina (Dual index primer set 1,NEB, E7600S) and the NEB library preparation kit for Illumina (NEB, #E7645S) were used for library preparation as previously described (Kalhor et al., 2017).
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description 520 Spleen-1
mutation_count_matrix_520.txt
Data processing Library strategy: evolving barcode
Illumina Nextseq 500 automatically used bcl2fastq2 (version 2.17) for basecalling. FASTQ files were downloaded from BaseSpace. Only the R1 files were used in the following analysis and only the R1 files were submitted.
To identify the barcoding region, we globally aligned each distinct sequence to the A26 reference barcode using TraceQC package in R. The parameters used for alignment are: 2 for match score, -2 for mismatch score, -6 for gap opening penalty and -0.1 for gap extension penalty.
The adapter sequences were trimmed off and sequences with less than 200 alignment score or less than 10 counts were filtered out from the subsequent analysis. The barcode sequence is 117 bp starting from 58 bp before predicted TSS.
The mutation events were categorized by the TraceQC package on each barcode into 4 attributes: 1) the mutation type (insertion, deletion or point mutation), 2) the starting position of mutation, 3) the length of mutation, and 4) the altered sequence.
The mutation events were normalized by the read count per million (RPM) approach and used to generate the feature count matrix for all the samples.
Supplementary_files_format_and_content: Processed data files are txt files including the count (RPM) of each mutation event.The mutation events are named in the form of A_B_C_D using below criteria: A, type of mutation, including insertion, deletion, or single nucleotide mutation (‘mutation’ in short); B, the starting position of mutation; C, the length of mutation; D, the altered sequence. For deletion, the value is -; for mutation, the value is the replaced nucleotide; for insertion, the value is the inserted sequence.
 
Submission date Nov 09, 2020
Last update date Apr 29, 2021
Contact name Igor L. Bado
E-mail(s) bado@bcm.edu
Organization name Baylor College of Medicine
Department Breast Center
Lab Xiang Zhang
Street address One Baylor Plaza, Alkek Building N1130
City HOUSTON
State/province Texas
ZIP/Postal code 77030
Country USA
 
Platform ID GPL19057
Series (2)
GSE161145 Bone invigorates metstatic seeding [evolving barcode]
GSE161146 Bone invigorates metastatic seeding
Relations
BioSample SAMN16711833
SRA SRX9464546

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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