|
| Status |
Public on Oct 28, 2016 |
| Title |
PA1_WT_IP_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
PA1_WT
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PA1
cell type: Embryonal carcinoma cells
transgene: FLAG-LIN28 (WT)
rip antibody: FLAG M2 (Sigma #A2220)
|
| Growth protocol |
PA1 cells (ATCC# CRL-1572) were maintained in DMEM/10% FBS.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were harvested in cold PBS and immediately lysed in M2 lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing 100 U/ml RNasin (Promega) and 2X protease and phosphatase inhibitors (Pierce). FLAG-tagged LIN28 variants were purified using the anti-FLAG M2 affinity gel following the manufacturer’s specifications (Sigma). Parental PA1 cells (no FLAG) were used as controls for antibody specificity. RNA was isolated from the beads using Trizol (Invitrogen) and RNeasy columns (Qiagen). Purified RNA was subjected to polyA selection using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB).
Libraries were prepared with the NEBNext Ultra RNA Library Prep Kit (NEB).
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
polyA+ RNA
RIP1_PA1_RPKM.xls
|
| Data processing |
From the raw reads, we removed adaptor sequences using a Cutadapt tool, allowing some mismatches (The specific parameters we used were -n 2 -e 0.1 -O 15 --quality-base=33).
Then, the cleaned reads were aligned to the human genome/transcriptome (hg19 and corresponding UCSC gene model) using a TopHat2 software with the following parameters (--library-type=fr-firststrand --min-intron-length=10 --no-coverage-search --microexon-search --min-isoform-fraction=0).
To estimate expression levels for each gene, we counted aligned reads per gene and using a htseq-count tool.
And calculated CPM (Counts Per Million) values were converted to RPKM (Reads Per Kilobase per Million mapped reads) values corresponding to gene lengths.
Genome_build: hg19
Supplementary_files_format_and_content: excel, RPKM values of genes after corresponding immunoprecipitation
|
| |
|
| Submission date |
Jun 30, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Areum Han |
| E-mail(s) |
areum.han@childrens.harvard.edu
|
| Organization name |
Boston Children's Hospital
|
| Lab |
Daley lab
|
| Street address |
Karp Family Building, 7th Floor. 300 Longwood Ave.
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE83905 |
RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT) or phospho-mimetic (S200D) FLAG-LIN28A in PA1 cells |
| GSE83906 |
RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT), phospho-mimetic (S200D), or phospho-null (S200A) FLAG-LIN28A |
|
| Relations |
| BioSample |
SAMN05331287 |
| SRA |
SRX1888935 |
