GSM5642123: cDNA_BR3_Tfcp2l1_Upstream; Mus musculus; OTHER
For iPCR samples: the combination of barcode and element(s) was excised from the plasmid by digestion with I-ceuI; this fragment was circularised; remaining linear fragments were destroyed; and circular fragments were linearised again with I-sceI. These linear fragments were amplified by PCR with sequencing adaptors. The final product was sequenced on an Illumina MiSeq platform using 150 bp paired-end reads. This process was done separately for each of the libraries. For mor details see van Arensbergen et a., 2017 (PMID: 28024146). For pDNA samples: For each library 1 µg of plasmid was digested with I-sceI in order to linearise the plasmid. Then, barcodes were amplified by PCR from 50 ng of material using the same primers and reaction conditions as in the amplification of cDNA in the Upstream assay, but only 9 cycles of amplification were used (1 min 96 °C, 9×(15 s 96 °C, 15 s 60 °C, 15 s 72 °C)). For each library, two technical replicates were carried out by using different index primers for each replicate. Samples were sequenced on an Illumina 2500 HiSeq platform with 65bp single end reads. For cDNA samples: RNA was extracted and processed for sequencing as described {van Arensbergen, 2017, 28024146} with a few modifications. Cells were harvested 24 h after transfection, resuspended in Trisure (#BIO-38032; Bioline) and frozen at -80 °C until further processing. From the Trisure suspension, the aqueous phase containing the RNA was extracted and loaded into RNA extraction columns (#K0732, Thermo Scientific). Total RNA was divided into 10 µl reactions containing 5 μg of RNA and was treated for 30 mins with 10 units of DNAse I (#04716728001; Roche). Then DNAse I was inactivated by addition of 1 ul of 1 μl of 25 mM EDTA and incubation at 70°C for 10 min. For the Upstream Assay the cDNA was produced and amplified by PCR as described {van Arensbergen, 2017, 28024146}. Per biological replicate 8 to 10 reactions were carried  in parallel in order to cover enough barcode complexity of the library. For the Downstream Assay the RNA was extracted and processed the same way until cDNA production. Here, cDNA was produced using a specific primer. Then cDNA was amplified in 2 steps (nested PCRs) in order to make the reaction strand-specific. The first PCR reaction run for 10 cycles (1 min 96 °C, 10×(15 s 96 °C, 15 s 60 °C, 15 s 72 °C)) using primers (index variants of) 285JvA (containing the S2, index and p7 adaptor) and 305JvA. Each 20 μl RT reaction was amplified in a 100-μl PCR reaction with MyTaq Red mix. The second PCR reaction was performed using 10ul of the product of the previous reaction in 100 μl reactions  (1 min 96 °C, 8×(15 s 96 °C, 15 s 60 °C, 15 s 72 °C)) using the same index variant primer and primer 437JvA (containing the S1, and p5 adaptor). For both Upstream and Downstream assays, the resulting PCR products were sequenced on an Illumina 2500 HiSeq platform with 65bp single end reads. PCR
GEO Accession
GSM5642123