This PCR product was then subcloned into pGEMT-Easy and the proper insert verified by EcoR I, Xho I and Sma I digestion.
Quantitative RT-PCR time-course analyses of four of these genes (KLK3, PYGB, TGM2 and SERINC3) (Figure 2 insert) verified both the Illumina expression data and the choice of the 16-hour time point after DHT treatment.
The PCR product was cloned into pGVB2 vector via SacI and NcoI sites and the insert verified by sequencing.
At least two inserts were verified by PCR for each 'element' except the Chr 6 element (only one insert verified) and the Chr 3 element (not verified but described by Tubio et al. [ 13]).
Three colonies were screened for each construct and the presence of insert verified by PCR using a vector specific forward primer and hCNTF gene specific reverse primer (Additional file 1: Figure S2).
The fragment was cloned into the pCR®T7 TOPO®-NT vector (Invitrogen Ltd., Paisley, UK) and used to transform E. coli BL21 (DE3) pLysS cells (Invitrogen Ltd., Paisley, UK); colonies were selected and inserts verified by sequencing.
Plasmid DNA was purified from cells with large barcode inserts, verified by digestion with BamH I, and sequenced with primer TAIL-LB LOX71 to determine the barcode sequences.
The Chr8 element showed both transduction and mapping to the native L1 insert of the two inserts verified, one had 3' unique sequence transduced and one was pure L1 3' terminus Transduced sequence, maximum extent of unique sequence observed in inserts verified by PCR (Additional file 2), except for Chr 3 element where read map position is given.
Correctness of the DNA insert was verified by sequencing (Microsynth AG, Balgach, Switzerland).
The single-cell-derived clones of viable cells containing the insert, as verified by RT-PCR, were expanded.
