Ribosome-associated RNAs (RPL-tag) processing

The co-immunoprecipitated ribosome-associated mRNA, using a tagged ribosomal protein Rpl22, was subjected to high throughput sequencing library preparation. To prepare the ribosome-associated mRNA library, TruSeq total RNA sample preparation protocol was followed according to manufacturer’s instructions. In brief, 0.25 to 0.5 ug of total RNA was diluted with nuclease-free water to a final volume of 5 ul in a PCR plate labeled as BRP. 2.5 ul of rRNA binding buffer and 2.5 ul of Ribo-Zero rRNA removal mix was added to each well. The plate was sealed and samples were incubated at 68^oC for 5 minutes and cooled down to 4^oC. The plate was incubated for 1 minute at room temperature. In a fresh PCR plate, 17.5 ul of rRNA removal beads were added to each well and 10 ul of the sample from the BRP plate was added. After mixing correctly by pipetting, the plate was incubated for 1 minute at room temperature and placed on the magnetic stand at RT for 1 minute. The supernatant was transferred to the corresponding well into a new plate. The new plate was again placed on the magnetic stand and supernatant was transferred to a new fresh plate. This procedure was repeated until all of the beads were removed.

After that, 49.5 ul of well-mixed RNAClean XP beads were added to each well and mixed by

pipetting. The plate was incubated at room temperature for 15 minutes and placed on the magnetic stand for 5 minutes at room temperature. The supernatant was removed, and beads were washed with 200 ul of 70% ethanol by incubating 30 seconds at room temperature and then discard the supernatant. The plate was air dried for 15 minutes at RT and then 5.5 ul Elution buffer was added and mixed properly. The plate was incubated at RT for 2 minutes and then placed on the magnetic stand for 5 minutes at RT. 4.25 ul of the supernatant was transferred to a new 0.3 ml PCR plate. In the new plate, 4.25 ul of Elute, prime, fragment high mix was added in each well and mixed by pipetting. The solution was incubated at 94^oC for 8 minutes in a thermal cycler. The plate was cooled to 4^oC and briefly centrifuged.

4.2.3.1 Synthesizing the first strand of cDNA

To reverse transcribe the cleaved RNA fragments that were primed with random hexamers into first strand cDNA, the following process was done. In the PCR plate, 4 ul of First strand synthesis Act D and SuperScript II mix was added and mixed properly. The plate was sealed and incubated at 25^oC for 10 minutes, 42^oC for 15 minutes, 70^oC for 15 minutes, 4^oC hold in a thermal cycler.

4.2.3.2 Synthesizing the second strand of cDNA

Following process was used to remove the RNA template and to synthesize a replacement strand, incorporating dTTP in place of dUTP to generate ds cDNA. AMPure XP beads were used to separate the ds cDNA from the second strand reaction mix. At the end of this process, blunt-ended cDNA was generated. To proceed with, 2.5 ul of Resuspension buffer and 10 ul of Second strand master mix was added to the samples and mixed properly. Samples were incubated at 16^oC for 1 hour and after that 45 ul of AMPure XP beads were added at the RT and mixed properly.

The plate was incubated at RT for 15 minutes and after that placed on the magnetic stand for 5 minutes. The supernatant was discarded, and beads were washed with 200 ul of 80% ethanol. The plate was incubated at RT for 30 seconds, and the supernatant was discarded. Ethanol washes were repeated two more times. The plate was dried at RT for 15 minutes, and 8.75 ul of Resuspension buffer was added and mixed properly. The plate was incubated at RT for 2 minutes

and again placed on the magnetic stand for 5 minutes at RT. 7.5 ul of the supernatant was transferred to a new 0.3 ml PCR plate (ALP plate).

4.2.3.3 Adenylation of 3’ end

A single ‘A’ nucleotide was added to the 3’ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single ‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensures a low rate of chimera (concatenated template) formation. For this reaction, 1.25 ul of Resuspension buffer and 6.25 ul of A-Tailing mix was added to the samples and mixed by pipetting. The plate was sealed and incubated at 37^oC for 30 minutes; 70^oC for 5 minutes and was held at 4^oC in a thermal cycler.

4.2.3.4 Adapters ligation

Multiple indexing adapters were ligated to the ends of the ds-cDNA to enable them to hybridize onto the flow cell. To the samples, 1.25 ul of the Resuspension buffer, 1.25 ul of Ligation mix and 1.25 ul of RNA adapter index was added and mixed by pipetting. The plate was sealed and incubated at 30^oC for 10 minutes. 2.5ul of Stop ligation buffer was added and mixed properly by pipetting. To the samples, 21 ul of the AMPure XP beads were added, and purification was done as described previously. At the end, 26.25 ul of the Resuspension buffer was added and mixed properly to recover the DNA molecules. Samples were incubated at RT for 2 minutes. The plate was placed on a magnetic stand for 5 minutes, and 25 ul of supernatant was transferred to a new plate (CAP plate). With 1X AMPure XP beads, samples were purified and isolated in 10 ul Resuspension buffer.

4.2.3.5 Selective enrichment of DNA fragments

To selectively enrich DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library following step was done. The PCR was performed with a PCR primer cocktail that anneals to the ends of the adapters. The number of PCR cycles was kept a minimum to avoid skewed representation of the library. In the adapter-ligated samples from the

previous step, 2.5 ul of PCR primer cocktail and 12.5 ul PCR master mix was added. Samples were mixed properly, and the plate was sealed. In a thermal cycler samples were incubated at (98^oC for 30 sec; 11 cycles of [98^oC for 10 sec; 60^oC for 30 sec, 72^oC for 30 sec]; 72^oC for 5 minutes; 4^oC hold). Samples were cleared using 1X AMPure XP beads and resuspended in 16.25 ul Resuspension buffer. 15 ul clear supernatant was transferred to a new 0.3 ml PCR plate (TSP1).

Samples were measured by Nanodrop, Qubit, and Agilent Bioanalyzer DNA 1000 kit. Libraries were normalized to 10nM concentration using Tris-Cl 10mM, pH 8.5 with 0.1% Tween 20 and pooled for sequencing into Hiseq 2000 sequencer.