A method for constructing a sequencing library based on a single-stranded DNA molecule is provided comprising: (1) forming a poly(C)n tail at a 3′-terminus of the single-stranded DNA molecule, to obtain a single-stranded DNA molecule with the poly(C)n tail with n representing a number of base C, and n being an integer ranging from 5 to 30; (2) obtaining a double-stranded DNA molecule by using an extension primer based on the single-stranded DNA molecule with the poly(C)n tail, with the extension primer comprising a H(G)m unit at a 3′-terminus thereof, H being base A, base T or base C, m being a number of base G, and m being an integer ranging from 5 to 15; and (3) ligating an adapter to one terminus of the double-stranded DNA molecule remote from the H(G)m unit, and amplifying the resulting ligation product to obtain an amplification product forming the sequencing library.

A method for constructing a sequencing library based on a single-stranded DNA molecule is provided comprising: (1) forming a poly(C)n tail at a 3′-terminus of the single-stranded DNA molecule, to obtain a single-stranded DNA molecule with the poly(C)n tail with n representing a number of base C, and n being an integer ranging from 5 to 30; (2) obtaining a double-stranded DNA molecule by using an extension primer based on the single-stranded DNA molecule with the poly(C)n tail, with the extension primer comprising a H(G)m unit at a 3′-terminus thereof, H being base A, base T or base C, m being a number of base G, and m being an integer ranging from 5 to 15; and (3) ligating an adapter to one terminus of the double-stranded DNA molecule remote from the H(G)m unit, and amplifying the resulting ligation product to obtain an amplification product forming the sequencing library.

Methods and systems for the high throughput RNA profiling or sequencing analysis of fixed single cells are provided. The methods and systems utilize barcoded microcarriers comprising a small carrier bead attached to a plurality of molecular barcodes with cleavable linkers, wherein these barcoded microcarriers are then combined in a system (e.g., a sealed system) with a single cell. The molecular barcodes are then released from the carrier beads to anneal to the mRNAs in the cells. Using reverse transcription, barcoded cDNAs is then generated from the mRNAs and the cells are lysed or digested to release the barcoded cDNAs for sequencing.

Methods and systems for the high throughput RNA profiling or sequencing analysis of fixed single cells are provided. The methods and systems utilize barcoded microcarriers comprising a small carrier bead attached to a plurality of molecular barcodes with cleavable linkers, wherein these barcoded microcarriers are then combined in a system (e.g., a sealed system) with a single cell. The molecular barcodes are then released from the carrier beads to anneal to the mRNAs in the cells. Using reverse transcription, barcoded cDNAs is then generated from the mRNAs and the cells are lysed or digested to release the barcoded cDNAs for sequencing.

Methods, compositions and kits for capturing, detecting and quantifying mature small RNAs are provided herein. Embodiments of the methods comprise tailing both the 5′ and 3′ ends of mature small RNA by ligating a 5′ ligation adaptor to the 5′ end and polyadenylating the 3′ end. Other embodiments comprise reverse transcribing the adaptor ligated, polyadenylated mature small RNA with a universal reverse transcription primer and amplifying the cDNA with universal primers.

Methods, compositions and kits for capturing, detecting and quantifying mature small RNAs are provided herein. Embodiments of the methods comprise tailing both the 5′ and 3′ ends of mature small RNA by ligating a 5′ ligation adaptor to the 5′ end and polyadenylating the 3′ end. Other embodiments comprise reverse transcribing the adaptor ligated, polyadenylated mature small RNA with a universal reverse transcription primer and amplifying the cDNA with universal primers.

Disclosed herein are methods for counting nucleic acid molecules (e.g., RNA molecules) of a sample by randomly truncating the nucleic acid molecules at a truncation base position within the nucleic acid molecules to produce truncated nucleic acid molecules, amplifying and sequencing the truncated nucleic acid molecules to produce sequencing reads, aligning the sequencing reads to a reference sequence to produce aligned sequencing reads, and identifying a number of nucleic acid molecules using truncation locations of aligned sequencing reads. Also disclosed herein are methods for constructing sequencing libraries that preserve truncation positions of the nucleic acid molecules. Also disclosed herein are methods for depleting or enriching a sample for one or more target sequences, using sets of blocking oligonucleotides corresponding to the one or more target sequences.

Disclosed herein are methods for counting nucleic acid molecules (e.g., RNA molecules) of a sample by randomly truncating the nucleic acid molecules at a truncation base position within the nucleic acid molecules to produce truncated nucleic acid molecules, amplifying and sequencing the truncated nucleic acid molecules to produce sequencing reads, aligning the sequencing reads to a reference sequence to produce aligned sequencing reads, and identifying a number of nucleic acid molecules using truncation locations of aligned sequencing reads. Also disclosed herein are methods for constructing sequencing libraries that preserve truncation positions of the nucleic acid molecules. Also disclosed herein are methods for depleting or enriching a sample for one or more target sequences, using sets of blocking oligonucleotides corresponding to the one or more target sequences.

Disclosed are nucleic acid-based molecular switches that respond to changes in pH. The switches may be used in DNA nanodevices. The switches may also act as sensors for measuring the pH of a sample, including cells, regions thereof, and whole organisms. The switch includes an A-motif that forms at acidic pH. Also disclosed are compositions and methods for measuring the pH of cells or regions thereof, such as vesicles, the nucleus, mitochondrial matrix, or the Golgi lumen.

Disclosed are nucleic acid-based molecular switches that respond to changes in pH. The switches may be used in DNA nanodevices. The switches may also act as sensors for measuring the pH of a sample, including cells, regions thereof, and whole organisms. The switch includes an A-motif that forms at acidic pH. Also disclosed are compositions and methods for measuring the pH of cells or regions thereof, such as vesicles, the nucleus, mitochondrial matrix, or the Golgi lumen.