Genome-wide detection and quantitation of RNA distribution by ChIRC 13a -seq

Eukaryotic genome are transcribed extensively, but a majority of transcripts remain functionally uncharacterized. This is most ascribed to lacking of a potent RNA-centric technology that is capable of accurately quantitating putative genomic binding sites for endogenous RNAs. We describe here a detailed protocol for Chromatin Isolation by RNA-Cas13a Complex sequencing (ChIRC 13a -seq), based on recently discovered CRISPR-Cas13a from Leptotrichia wadei (LwaCas13a), for proling of RNA associated chromatin binding cites. ChIRC 13a -seq employs biotinylated, enzymatically-dead Cas13a (dCas13a) that is still capable of binding target RNA and guide RNAs (gRNAs) specic for the RNA target of interest to enrich RNA and its chromatin binding sites. This assay can be performed in standard molecular biology laboratories with 2 d taken for ChIRC 13a -seq library preparation. binding prole or few single guide RNAs for each target RNA 8,9 CRISPR-Cas13a demonstrates to target RNA than oligonucleotide probes base-pairing with target ChIRP based approaches, shows more applicability on of very long noncoding RNA associated genomic DNA binding prole, experimental perspectives. present here a step-by-step protocol for ChIRC 13a -seq.


Introduction
Long noncoding RNAs (lncRNAs) have been increasingly reported to play critical roles in a broad range of biological processes 1-4 , including transcription 5 , but our understanding of lncRNAs-mediated gene regulation is still largely elusive. A comprehensive understanding of genome-wide RNA associated chromatin binding pro le would therefor provide key insights into the molecular mechanism by which lncRNAs exert their roles in controlling gene regulation.
Currently, chromatin isolation by RNA puri cation sequencing (ChIRP-seq) is the gold-standard method for quantitative interrogation of RNA associated chromatin pro le 6 , although the development of other oligonucleotides probe based base-paring methods such as RAP and CHART-seq approaches has permitted successful mapping of several RNAs 4,7 . In ChIRP approach, a large number of probes for the tiling of an entire transcript are required to achieve robust enrichment of target RNA, with the concomitant increased risk of potential off-target effects that may be generated by the pool of probes used in the assay, especially for very long noncoding transcripts. ChIRC 13a -seq addresses this challenge by enabling reproducible detection and quantitation of RNA associated chromatin binding pro le with only one or few single guide RNAs used for each target RNA 8,9 . Considering CRISPR-Cas13a demonstrates more speci city and a nity to target RNA than oligonucleotide probes (via base-pairing with target RNA) used in ChIRP based approaches, ChIRC 13a -seq shows more potential applicability on examination of very long noncoding RNA associated genomic DNA binding pro le, both from economic and experimental perspectives. We present here a step-by-step protocol for ChIRC 13a -seq. 2. Remove residual PBS and add 10 ml fresh 1% Formaldehyde Solution (Formaldehyde in PBS) to each 10 cm plate. Swirl plates brie y and incubate at room temperature for 15 minutes.

Reagents
3. Add 1 ml of 1.25 M glycine to plates. Swirl plates brie y and incubate at room temperature for 5 minutes to quench formaldehyde. 4. Rinse cells three times with 5 ml PBS for each plate. Add 1 ml ice cold PBS to each 10 cm plate and then scape out cells by using silicon scraper. 5. Harvest cells in 1.7 ml Eppendorf tube for each 10 cm plate and spin at 5,000 g for 5 minutes at 4°C. 6. Discard supernatant, resuspend pellet in 1 ml ice cold PBS per 5x10 7 cells and spin at 5000g for 5 minutes at 4°C. 2. Samples were sheared using a Bioruptor® Pico sonication device using the following parameters: Cycle = 5, On =30s, Off = 30s, Time = 5 min. (Note: sonication condition may need to be adjusted for different sonication devices, cell types and cell numbers used.) After sonication, we expect to see sheared DNA that ranges from 100 -500 bp in size.
3. Spin at 20,000g for 15 min at 4°C to pellet debris. F. Wash, elution, and cross-link reversal 1. Collect the beads using magnetic rack. Remove supernatant and wash beads for 5 min with 2% SDS in TE buffer (10 mM Tris, pH 8, 1 mM EDTA). Collect the beads using magnetic rack. Repeat this wash one more time.
2. Collect the beads using magnetic rack. Remove supernatant and wash beads for 5 min with high salt buffer (50 mM HEPES pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate and 500 mM NaCl). Collect the beads using magnetic rack. Repeat this wash one more time.
4. Collect the beads using magnetic rack. Remove supernatant and wash beads once with 1 ml TE that contains 50 mM NaCl.
5. Spin at 20,000g for 1 min and collect the beads using magnetic rack to remove residual TE buffer.
6. Add 2.5 ul Proteinase K. Incubate beads for 2 h at 55 °C and then overnight at 65 °C with mixing to decrosslink. The same procedure is followed for input samples including RNase and proteinase K digestion. 7. After overnight de-crosslinking, purify DNA by QIAquick Spin columns.
Library preparation 1. Library is prepared by NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® according to the instructions provided by the manufacturer. However, there are many other options, as long as the chosen kit is applicable to deep sequencing with the Illumina's system.

Bioinformatics analysis
The bioinformatics analysis pipeline of ChIRC-seq data included the following steps : a) Quality control : FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and TRIMMOMATIC (http://www.usadellab.org/cms/?page=trimmomatic) were used to inspect the quality of the sequencing reads, and to trim the sequencing adaptors, respectively. b) Read alignment : Bowtie2 was used to align ChIRC-seq samples to hg38 genome sequence. After the sequence alignment, only 1 copy/read per genome position was kept for downstream analysis. c) Peak nding : The ChIRC13a-seq peaks were called by using HOMER ndPeaks subroutine (http://homer.ucsd.edu/homer/). Another HOMER script annotatePeaks.pl was used in order to annotate the peaks and to estimate the raw or normalized counts within the peak regions. d) Generation of bedgraph les: The HOMER scripts makeUCSC le and makeMultiWigHub.pl were used in order to generate bedgraph les for the visualization of ChIRC-seq data in the UCSC genome browser.