After transfer to ice-cold Trizol, hippocampi were homogenized using a tissue homogenizer (Salm&Kipp, Breukelen, The Netherlands) and total RNA was isolated according to the manufacturer’s protocol. After precipitation, RNA was purified with Qiagen’s RNeasy kit with on-column DNase digestion.
Library strategy
OTHER
Library source
transcriptomic
Library selection
other
Instrument model
Illumina Genome Analyzer
Description
Sequence tag preparation was done with Illumina’s Digital Gene Expression Tag Profiling Kit according to the manufacturer’s protocol (version 2.1B). A schematic overview of the procedure is given in the figure at the end of this document. One microgram of total RNA was incubated with oligo-dT beads to capture the polyadenlyated RNA fraction. First and second strand cDNA synthesis were performed while the RNA was bound to the beads. While on the beads, samples were digested with NlaIII to retain a cDNA fragment from the most 3’ CATG to the polyA-tail. Subsequently, the GEX adapter 1 was ligated to the free 5’ end of the RNA, and a digestion with MmeI was performed, which cuts 17 bp downstream of the CATG site. At this point, the fragments detach from the beads. After dephosphorylation and phenol extraction, the GEX adapter 2 was ligated to the 3’ end of the tag. A PCR amplifcation with 15 cycles using Phusion polymerase (Finnzymes) was performed with primers complementary to the adapter sequences to enrich the samples for the desired fragments. The resulting fragments of 85 bp were purified by excision from a 6% polyacrylamide TBE gel. The DNA was eluted from the gel debris with 1x NEBuffer 2 by gently rotation for 2h at room temperature. Gel debris were removed using Spin-X Cellulose Acetate Filter (2 ml, 0.45 µm) and the DNA was precipitated by adding 10 µl of 3M sodium acetate (pH 5.2) and 325 µl of ethanol (-20C), followed by centrifugation at 14,000 rpm for 20 minutes. After washing the pellet with 70% ethanol, the DNA was resuspended in 10 µl of 10 mM Tris-HCl,. pH8.5 and quantified the DNA with a Nanodrop 1000 spectrophotometer.
Data processing
Cluster generation was performed after applying 4 pM of each sample to the individual lanes of the Illumina 1G flowcell. After hybridization of the sequencing primer to the single stranded products, 18 cycles of base incorporation were carried out on the 1G analyzer according to the manufacturer’s instructions. Image analysis and basecalling was performed using the Illumina Pipeline, where sequence tags were obtained after purity filtering. This was followed by sorting and counting the unique tags.
