cDNA's were digested with NLA III to anchor the sequence tag to the 3'most NLA III site, after which a tagging adaptor containing an Mme1 recogniton site, and a SOLiD system PCR adaptor sequence, was ligated to the bead-attached cDNA.
Insertion of a 6xHis-tag into the predicted loops, including βB-βC, βD-βE, βF-βG and βH-βI (Fig. 1), was performed using PCR mediated mutagenesis, to introduce SpeI and SstI restriction sites, and subsequent cloning of a His-tag adaptor into the loops.
After removal of the low-quality tags, adaptor tags, and one copynumber tag, a total of 4,841,402 and 5,395,715 clean tags were obtained from the two libraries with 100,107 and 108,572 unique nucleotide sequences, respectively (Additional file 2, Table S2).
The purified RNAs were then dephosphorylated by bacterial alkaline phosphatase (BAP) treatment and a biotin tagged 3' adaptor (5' phosphorylated) (Additional file 5 Table S8) was ligated to the RNA molecules by T4 RNA ligase (Promega, Shanghai, China).
However, increasing the sample size to thousands necessitates additional tagged adaptors leading to preparation of thousands of libraries, which is time consuming and expensive.
After digestion, the 21 bp unique tag and adaptor were purified, dephosphorylated, phenol extracted and ligated to the GEX adaptor 2, complementary to the surface-bound amplification primer on the flow cell.
Next, the library was immobilized onto streptavidin beads, facilitated by a 5' biotin tag on Adaptor B. Finally, the unbound strand of each fragment (with 5'-Adaptor A) was released, and the quality of the recovered single-stranded DNA library was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies).
Interfering with a putative synaptic tag, the adaptor protein KIBRA, which protects the atypical PKM from degradation, selectively erases associative LTF.
Next, the library was immobilized onto streptavidin beads, facilitated by a 5' biotin tag on Adaptor B, and any nicks in the double-stranded library are repaired.
Using custom scripts, raw sequence files in the QSEQ format [ 23] for an individual were scanned for any barcode tag, sequencing adaptor, and enzyme cut site sequence and these were trimmed from the sequence ends.
Clean tags were obtained after filtering the adaptor tags, empty tags, low-quality tags, and one-copy tags from the total tags, and then they were aligned to the reference database and annotated.
