GSM4848155: SSD1_3_42_CRAC; Saccharomyces cerevisiae BY4741; OTHER
The full protocol for extraction of RNAs bound to SSD1 is available on protocols.io: http://dx.doi.org/10.17504/protocols.io.5ppg5mn. Briefly, total cell lysates were extracted from the frozen cell pellets using Zirconia beads, cleared of cell debris and then incubated with IgG Sepahrose beads to pull down the ProteinA moiety of the HTP-tagged SSD1 and its cross-linked RNAs. After sufficient washes to remove non-specific protein, etc, the bound protein was released from the beads using HALO-TEV protease and then collected by passing through SnapCap columns.The cross-linked RNA was partially digested with a 1:100 dilution of RNace-IT (Promega) at 37oC for 5 min and stopped by the addition of Guanidine HCl. NaCl and Imidazole were added to 300mM and 10mM respectively  and the protein/RNA mixtures bound to Nickel beads at 4oC overnight. The beads were transferred to fresh SnapCap columns, washed extensively and the cross-linked RNAs treated with Alkaline Phosphatase to remove 3' phosphates left after RNase-cleavage. After extensive washing and removal of Guanidinium, a miRCat-33 linker (activated 5' and blocked 3') was ligated to the 3'-end for 5h at 25oC. After further washing, the 5'ends of the bound RNAs were phosphorylated using T4 Polynucleotide Kinase and gamma-32P-ATP, adding cold ATP to ensure almost all of the 5' ends were phosphorylated, and the columns extensively washed again. 5' SOLEXA linkers (mixed DNA:RNA oligos with inverted ddT at the 5' end to prevent degradation, a barcode sequence at the 3' end  and a random 3mer to reveal PCR duplicates plus constant 3 nucleotide 3' termini to ensure all linkers ligate with equal efficiency) were ligated on-bead and incubated at 18oC overnight. The beads were washed 3x and the tagged protein-RNA samples eluted by increasing the Imidazole concetration to 150mM. 30 and 42oC samples from the same biological replicate were pooled at this stage and the mixture was precipitated with 5V acetone, in the presence of 40ug GlycoBlue to help visualise the pellets, by incubation at -20oC for 2 hours.The pellets were resuspended in 1x NuPAGE Sample Buffer, heated to 65oC for 10 mins and loaded and run on a 10 well NuPAGE 4-12% gradient gel alongside SeeBlue Plus 2 Markers in 1x MOPS Buffer which was then blotted onto Hybond C Extra Nitrocellulose membrane in 1x NuPAGE Transfer Buffer (without methanol as SSD1-HTP is a big protein). The membrane was briefly dried and placed between 2 acetate sheets and exposed for autoradiography with intensifying screens at -80oC overnight. After developing the film, strong bands with smears above of protein:RNA complexes of the expected sizes (around 145kDa) in the SSD1-HTP sample lanes but only a faint smear in the BY4741 sample were detected. The film was used to identify the location of the smears on the membrane and cut through to remove the relevant fragments of nitrocellulose which were placed in Eppendorf SafeLock tubes and then digested with Proteinase K for 2h at 55oC before Phenol/Chloroform/Isoamyl alcohol extraction, ethanol precipitation in the presence of 20ug of Glycoblue, washed in 70% ethanol and resuspended in Reverse Transcription (RT) Reaction Mix containing miRCat oligo and dNTPs, heated at 80oC for 3 min and Superscript IV Reverse Transcriptase, RNasin and DTT added to the reactions which were incubated at 50oC for  1 hour. The enzyme was heat inactivated andthe samples digested with RNase H for 30 mins at 37oC before storing at -20oC overnight. 3x 50ul PCR Reactions for each of the 3 RT reactions were prepared with PCRFwd and Rev primers, LA Taq Buffer, dNTPS and LA Taq Polymerase and amplified  for 22 cycles to yield 5-50ng  of DNA from each sample. After pooling the PCR reactions, ethanol precipitation and resuspending  in 1x Purple Loading Dye (NEB), the amplified samples were run in alternating lanes on a 3% Metaphor agarose gel in 1x TBE Buffer with 50bp ladders run on either side and staining with SYBRSafe. The gel was then scanned on a PhosphorImager using the '1 laser option' and the negative image printed at 100% magnification and used as a template to determine the areas of the gel containing the amplified library samples  (larger than primer dimers and <180bp fragments) which were then excised by careful cutting with a scalpel, extracted from the gel using a Qiagen Gel Extraction Kit, washing an extra time to ensure complete removal of Guanidine HCl.  20ul of nuclease-free water was added to each column and left on the membrane for 5 mins before elution. Individual library samples (+/- HS) were quantified on the Qubit and, assuming an average DNA Fragment size of 150nt (including linkers) diluted to 10nM before mixing 1ul of each library sample, making up to 5ul with nuclease-free water, adding 5ul of 0.1M NaoH and denaturing the combined library for 5 mins at room temperature, neutralising with 5ul of 200mM Tris.HCl, ph6.8, vortexing and spinning down before mixing with 985ul of of Illumina Hybridization Buffer, vortexing and spinning again. 500ul of this pooled library  was then loaded on the Illumina MiniSeq following the manufacturer's instructions.