 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 23, 2020 |
| Title |
OCM1A_BAP1-HyPBase |
| Sample type |
SRA |
| |
|
| Source name |
OCM-1A
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: OCM-1A
tissue: uveal melanoma
|
| Treatment protocol |
We transfected 10 replicates of OCM-1A cells 1.25 µg of PB-SRT-Puro and 1.25 µg of either HyPBase or BAP1-HyPBase (gifts from Dr. Michael Onken) using the TransIT-LT1 transfection reagent following manufacturer’s protocol for 6-well plates. Puromycin was added after 24 hours to a final concentration of 2 µg/ml. Cells were grown under selection for one week, by which time almost all negative control transfectants were dead. After 7 days, we dissociated each replicate with trypsin-EDTA and created single cell suspensions in phosphate-buffered saline (PBS) Aliquots of each replicate were cryopreserved in cell culture media (see above) supplemented with 5% DMSO. The remaining cells were pelleted by centrifugation at 300g for 5 minutes. Cell pellets were either processed immediately or kept at -80ºC in RNAProtect Cell Reagent.
|
| Growth protocol |
Cells were cultured in RPMI 1640 Medium supplemented with 10% fetal bovine serum (FBS) and 1% of either penicillin-streptomycin or antibiotic-antimycotic. Cells were grown at 37ºC with 5% carbon dioxide (CO2). Media was replenished every 2 days.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated from each replicate using the RNEasy Plus Mini Kit following manufacturer’s instructions. Briefly, cell pellets were resuspended in 600 µl of Buffer RLT Plus with 1% 2-mercaptoethanol. Cells were homogenized by vortexing. DNA was removed by running lysate through gDNA Eliminator spin columns, while RNA was bound by passing the flow-through over RNEasy spin columns. An on-column treatment with DNaseI was also performed. After washing, RNA was eluted in 40 µl RNase-free H2O. RNA was quantitated using the Qubit RNA HS Assay Kit. We performed first strand synthesis on each replicate with Maxima H Minus Reverse Transcriptase. We mixed 2 µg of total RNA with 1 µl 10 mM dNTPs and 1 µl of 50 µM SMART_dT18VN primer (for a complete list of oligonucleotides, see Table S5), brought the total volume up to 14 µl, and incubated it at 65ºC for 5 minutes. After transferring to ice and letting rest for 1 minute, we added 4 µl 5X Maxima RT Buffer, 1 µl RNaseOUT, and 1 µl of 1:1 Maxima H Minus Reverse Transcriptase diluted in 1x RT Buffer (100 U). The solution was mixed by pipetting and incubated at 50ºC for 1 hour followed by heat inactivation at 85ºC for 10 minutes. Finally, we digested with 1 µl RNaseH at 37ºC for 30 minutes. cDNA was stored at -20ºC.
The PCR conditions for amplifying self-reporting transcripts (i.e. transcripts derived from self-reporting transposons) involved mixing 1 µl cDNA template with 12.5 µl Kapa HiFi HotStart ReadyMix, 0.5 µl 25 µM SMART primer, and either 1 µl of 25 µM SRT_PAC_F1 primer (in the case of puromycin selection) or 0.5 µl of 25 µM SRT_tdTomato_F1 primer (in the case of tdTomato screening). The mixture was brought up to 25 µl with ddH2O. Thermocycling parameters were as follows: 95ºC for 3 minutes; 20 cycles of: 98ºC for 20 seconds–65ºC for 30 seconds–72ºC for 5 minutes; 72ºC for 10 minutes; hold at 4ºC forever. As a control, cDNA quality can be assessed with exon-spanning primers for β-actin (see Table S5 for examples of human primers (Raff et al., 1997)) under the same thermocycling settings. PCR products were purified using AMPure XP beads. 12 µl of resuspended beads were added to the 25 µl PCR product and mixed homogenously by pipetting. After a 5-minute incubation at room temperature, the solution was placed on a magnetic rack for 2 minutes. The supernatant was aspirated and discarded. The pellet was washed twice with 200 µl of 70% ethanol (incubated for 30 seconds each time), discarding the supernatant each time. The pellet was left to dry at room temperature for 2 minutes. To elute, we added 20 µl ddH2O to the pellet, resuspended by pipetting, incubated at room temperature for 2 minutes, and placed on a magnetic rack for one minute. Once clear, the solution was transferred to a clean 1.5 ml tube. DNA concentration was measured on the Qubit 3.0 Fluorometer using the dsDNA High Sensitivity Assay Kit. Calling card libraries from bulk RNA were generated using the Nextera XT DNA Library Preparation Kit. One nanogram of PCR product was resuspended in 5 µl ddH2O. To this mixture we added 10 µl Tagment DNA (TD) Buffer and 5 µl Amplicon Tagment Mix (ATM). After pipetting to mix, we incubated the solution in a thermocycler preheated to 55ºC. The tagmentation reaction was halted by adding 5 µl Neutralization Tagment (NT) Buffer and was kept at room temperature for 5 minutes. The final PCR was set up by adding 15 µl Nextera PCR Mix (NPM), 8 µl ddH2O, 1 µl of 10 µM transposon primer (e.g. OM-PB-NNN) and 1 µl Nextera N7 indexed primer. The transposon primer anneals to the end of the transposon terminal repeat–piggyBac, in the case of OM-PB primers, or Sleeping Beauty, in the case of OM-SB primers–and contains a 3 base pair barcode sequence. Every N7 primer contains a unique index sequence that is demultiplexed by the sequencer. Each replicate was assigned a unique combination of barcoded transposon primer and indexed N7 primer, enabling precise identification of each library’s sequencing reads. The final PCR was run under the following conditions: 95ºC for 30 seconds; 13 cycles of: 95ºC for 10 seconds–50ºC for 30 seconds–72ºC for 30 seconds; 72ºC for 5 minutes; hold at 4ºC forever. After PCR, the final library was purified using 30 µl (0.6x) AMPure XP beads, as described above. The library was eluted in 11 µl ddH2O and quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D1000 ScreenTape.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Bulk DNA calling cards library
SRRs represent biological replicates.
|
| Data processing |
Reads were demultiplexed by the N7 index sequences added during the final PCR. Read 1 began with the 3 base-pair barcode followed by the end of the transposon terminal repeat, culminating with the insertion site motif (TTAA in the case of piggyBac; TA in the case of Sleeping Beauty) before entering the genome. piggyBac reads were checked for exact matches to the barcode, transposon sequence, and insertion site at the beginning of reads before being hard trimmed using cutadapt (Martin, 2011) with the following settings: -g "^NNNTTTACGCAGACTATCTTTCTAGGGTTAA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels, where NNN is replaced with the primer barcode. Sleeping Beauty libraries were trimmed with the following settings: -g "^NNNTAAGTGTATGTAAACTTCCGACTTCAACTGTA" --minimum-length 1 --discard-untrimmed -e 0 --no-indels. Reads passing this filter were then trimmed of any trailing Nextera adapter sequence, again using cutadapt and the following settings: -a "CTGTCTCTTATACACATCTCCGAGCCCACGAGACTNNNNNNNNNNTCTCGTATGCCGTCTTCTGCTTG" --minimum-length 1. The remaining reads were aligned to the human genome (build hg38) with NovoAlign and the following settings: -n 40 -o SAM -o SoftClip. Aligned reads were validated by confirming that they mapped adjacent to the insertion site motif. Successful reads were then converted to calling card format (.ccf.txt; see http://wiki.wubrowse.org/Calling_card) using custom programs (available at https://github.com/arnavm/calling_cards) and visualized on the WashU Epigenome Browser v46 (Zhou et al., 2011) (http://epigenomegateway.wustl.edu/legacy/).
Genome_build: hg38
Supplementary_files_format_and_content: BAM files contain aligned and annotated calling card reads. ccf.txt files contain processed calling card data. describes calling card-specific BAM tags.
|
| |
|
| Submission date |
Apr 10, 2020 |
| Last update date |
Jul 23, 2020 |
| Contact name |
Robi D Mitra |
| E-mail(s) |
rmitra@wustl.edu
|
| Organization name |
Washington University in St. Louis
|
| Department |
Genetics
|
| Street address |
4515 McKinley Ave
|
| City |
St. Louis |
| State/province |
Missouri |
| ZIP/Postal code |
63143 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE148448 |
Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells |
|
| Relations |
| BioSample |
SAMN14573319 |
| SRA |
SRX8094407 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4471642_OCM1A-BAP1-HyPBase.bam |
5.1 Gb |
(ftp)(http) |
BAM |
| GSM4471642_OCM1A-BAP1-HyPBase.ccf.txt.gz |
36.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
