Probe-based analysis of nucleic acids and proteins

Abstract

Provided herein are systems and methods for processing biomolecules (e.g., nucleic acid molecules, proteins) from a sample. A method for processing biomolecules may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule) and barcoding the probe-nucleic acid molecule complex or derivatives thereof. Such a method can comprise performing a nucleic acid reaction, e.g., extension, denaturation, and amplification. A method for processing a sample may comprise hybridizing probes to (i) target regions of a nucleic acid molecule (e.g., RNA molecule) and (ii) a reporter oligonucleotide of a feature binding group, and barcoding the probe-associated molecules. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.

What is claimed is: 
     
         1 . A method for analyzing a nucleic acid molecule of an embedded, fixed tissue, comprising:
 (a) providing the embedded, fixed tissue, wherein:
 (i) the embedded, fixed tissue comprises a plurality of cells, 
 (ii) a cell of the plurality of cells comprises the nucleic acid molecule, and 
 (iii) the embedded, fixed tissue is embedded in a solid medium; 
   (b) removing at least a portion of the solid medium from the embedded, fixed tissue, thereby obtaining a fixed tissue comprising the plurality of cells;   (c) dissociating the fixed tissue into cells of the plurality of cells, wherein the cells comprise the cell comprising the nucleic acid molecule; and   (d) in the cell, using a barcode molecule to generate a barcoded nucleic acid molecule comprising a barcode sequence and a sequence corresponding to the nucleic acid molecule.   
     
     
         2 . The method of  claim 1 , wherein the fixed tissue is fixed at least about 1 year prior to (a). 
     
     
         3 . The method of  claim 2 , wherein the fixed tissue is fixed at least about 5 years prior to (a). 
     
     
         4 . The method of  claim 1 , further comprising, in (b), adding a first solvent to the embedded, fixed tissue, thereby dissolving the solid medium, thereby obtaining a tissue. 
     
     
         5 . The method of  claim 4 , wherein the first solvent is a non-polar solvent. 
     
     
         6 . The method of  claim 5 , wherein the non-polar solvent comprises xylene. 
     
     
         7 . The method of  claim 5 , wherein the non-polar solvent comprises Neo-Clear® xylene alternative. 
     
     
         8 . The method of  claim 4 , further comprising removing the first solvent from the tissue. 
     
     
         9 . The method of  claim 8 , further comprising adding a second solvent to the tissue. 
     
     
         10 . The method of  claim 9 , wherein the second solvent is a polar solvent. 
     
     
         11 . The method of  claim 10 , wherein the polar solvent comprises ethanol. 
     
     
         12 . The method of  claim 9 , further comprising removing the second solvent from the tissue. 
     
     
         13 . The method of  claim 12 , further comprising adding a rehydration agent to the tissue. 
     
     
         14 . The method of  claim 13 , wherein the rehydration agent comprises water. 
     
     
         15 . The method of  claim 13 , further comprising removing the rehydration agent from the tissue. 
     
     
         16 . The method of  claim 15 , further comprising adding buffer to the tissue. 
     
     
         17 . The method of  claim 16 , further comprising removing the buffer from the tissue. 
     
     
         18 . The method of  claim 17 , further comprising, in (c), dissociating the tissue. 
     
     
         19 . The method of  claim 18 , further comprising resuspending the tissue in a supernatant. 
     
     
         20 . The method of  claim 19 , further comprising filtering the tissue. 
     
     
         21 . The method of  claim 20 , further comprising washing the tissue. 
     
     
         22 . The method of  claim 21 , further comprising resuspending the tissue. 
     
     
         23 . The method of  claim 18 , wherein the dissociating the tissue is performed using an automated dissociator. 
     
     
         24 . The method of  claim 23 , wherein said automated dissociator is a gentleMACS™ Octo Dissociator. 
     
     
         25 . The method of  claim 18 , wherein the dissociating the tissue is performed using a manual pulverizer. 
     
     
         26 . The method of  claim 1 , wherein (b) and (c) provide a yield of cells of at least about 1×10 5  cells per two 25 micrometer sections of the fixed tissue. 
     
     
         27 . The method of  claim 1 , wherein the fixed tissue comprises fixed human tissue. 
     
     
         28 . The method of  claim 27 , wherein the fixed tissue comprises one or more of fixed connective tissue, fixed epithelial tissue, fixed organ tissue, fixed muscle tissue, fixed ligaments, fixed tendons, fixed skin tissue, fixed breast tissue, fixed bladder tissue, fixed kidney tissue, fixed liver tissue, fixed colon tissue, fixed thyroid tissue, fixed cervical tissue, fixed prostate tissue, fixed lung tissue, fixed cardiac tissue, fixed heart tissue, fixed muscle tissue, fixed pancreas tissue, fixed anal tissue, fixed bile duct tissue, fixed a bone tissue, fixed bone marrow, fixed uterine tissue, fixed ovarian tissue, fixed endometrial tissue, fixed vaginal tissue, fixed vulvar tissue, fixed stomach tissue, fixed ocular tissue, fixed nasal tissue, fixed sinus tissue, fixed penile tissue, fixed salivary gland tissue, fixed gut tissue, fixed gallbladder tissue, fixed gastrointestinal tissue, fixed bladder tissue, fixed brain tissue, fixed spinal tissue, fixed neurons, fixed cells representative of a blood-brain barrier, fixed blood, fixed hair, fixed nails, fixed keratin, or fixed collagen. 
     
     
         29 . The method of  claim 1 , wherein (d) comprises hybridizing the barcode molecule to the nucleic acid molecule. 
     
     
         30 . The method of  claim 29 , further comprising extending the barcode molecule to form the barcoded nucleic acid molecule.